Heat-stable carbonic anhydrases and their use

ABSTRACT

The present invention relates to use of heat-stable carbonic anhydrase in CO 2  extraction, e.g., from flue gas, natural gas or biogas. Furthermore, the invention relates to isolated polypeptides having carbonic anhydrase activity at elevated temperatures and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/004,066 filed Jan. 11, 2011, which is a continuation of U.S.application Ser. No. 12/849,446 (now U.S. Pat. No. 7,892,814) filed Aug.3, 2010 which is a continuation of U.S. application Ser. No. 12/523,975(now U.S. Pat. No. 7,803,575) filed Jul. 21, 2009 which is a 35 U.S.C.371 national application of PCT/US2008/052567 filed Jan. 31, 2008, whichclaims priority or the benefit under 35 U.S.C. 119 of Danish applicationno. PA 2007 00157 filed Jan. 31, 2007 and U.S. provisional applicationNo. 60/887,386 filed Jan. 31, 2007, the contents of which are fullyincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to use of heat-stable carbonic anhydraseat elevated temperature in CO₂ extraction, e.g., from flue gasses,biogas or natural gas. The invention also relates to bioreactors forextracting carbon dioxide. Furthermore, the invention relates toisolated polypeptides having carbonic anhydrase activity at elevatedtemperatures and isolated polynucleotides encoding the polypeptides, aswell as formulation of the polypeptide. The invention also relates tonucleic acid constructs, vectors, and host cells comprising thepolynucleotides.

BACKGROUND OF THE INVENTION

Carbonic anhydrases (CA, EC 4.2.1.1, also termed carbonate dehydratases)catalyze the inter-conversion between carbon dioxide and bicarbonate[CO₂+H₂O⇄HCO₃ ⁻+H⁺]. The enzyme was discovered in bovine blood in 1933(Meldrum and Roughton, 1933, J. Physiol. 80: 113-142) and has since beenfound widely distributed in nature in all domains of life. These enzymesare categorized in three distinct classes called the alpha-, beta- andgamma-class, and potentially a fourth class, the delta-class. Theseclasses evolved from independent origins (Bacteria, Archaea, Eukarya)and have no significant sequence or structural identity, except forsingle zinc atom at the catalytic site (for review see Tripp et al.,2001, J. Biol. Chem. 276: 48615-48618). For alpha-CAs more than 11isozymes have been identified in mammals. Alpha-carbonic anhydrases areabundant in all mammalian tissues where they facilitate the removal ofCO₂. Beta-CAs are ubiquitous in algae and plants where they provide forCO₂ uptake and fixation for photosynthesis. The gamma-class of CAs isbelieved to have evolved first. The only gamma-CA that has been isolatedand characterized so far is from the Archaeon Methanosarcina thermophilastrain TM-1 (Alber and Ferry, 1994, Proc. Natl. Acad. Sci. USA 91:6909-6913), however many gamma-type carbonic anhydrases have beenproposed by Parisi et al., 2004, Plant Mol. Biol. 55: 193-207. Inprokaryotes genes encoding all three CA classes have been identified,with the beta- and gamma-class predominating. Many prokaryotes containcarbonic anhydrase genes from more than one class or several genes ofthe same class (for review see Smith and Ferry, 2000, FEMS Microbiol.Rev. 24: 335-366; Tripp et al., 2001. J. Biol. Chem. 276: 48615-48618).

Carbon dioxide (CO₂) emissions are a major contributor to the phenomenonof global warming. CO₂ is a by-product of combustion and it createsoperational, economic, and environmental problems. CO₂ emissions may becontrolled by capturing CO₂ gas before emitted into the atmosphere.There are several chemical approaches to control the CO₂ emissions.However, many of these approaches have draw backs such as high energyconsumption, slow processes, and use of ecological questionable or toxiccompounds.

An enzyme based solution using the capability of carbonic anhydrase tocatalyse the conversion of CO₂ to bicarbonate at a very high rate(turnover is up to 10⁵ molecules of CO₂ per second), takes care of thespeed and environmental issues in relation to CO₂ capture. Technicalsolutions for extracting CO₂ from gases, such as combustion gases orrespiration gases, using carbonic anhydrases have been described in WO2006/089423, U.S. Pat. No. 6,524,842, WO 2004/007058, WO 2004/028667, US2004/0029257, U.S. Pat. No. 7,132,090, WO 2005/114417, U.S. Pat. No.6,143,556, WO 2004/104160, US 2005/214936. Generally, these techniquesoperate by bringing a soluble or immobilized carbonic anhydrase intocontact with CO₂ which either may be in a gas phase or a liquid phase.The carbonic anhydrase catalyses the conversion of CO₂ into bicarbonateand/or carbonate ions. The ions may either be utilized to facilitategrowth of algae or other microorganisms, to induce a pH change in asurrounding medium or supply buffering capacity, to providebicarbonate/carbonate as an active agent for subsequent chemicalprocesses, or precipitated as a carbonate salt, or converted back intopure CO₂, which can then be used (for example in enhanced oil recovery,for production of urea, for food and beverage processing, or to supplyCO₂ to greenhouses), released (for example from a contained life supportenvironment such as a submarine or spacecraft), compressed (for examplefor transportation through pipelines), or stored under compression (suchas in geological or deep oceanic formations).

Mammalian, plant and prokaryotic carbonic anhydrases (alpha- andbeta-class CAs) generally function at physiological temperatures (37°C.) or lower temperatures. The temperature of combustion gasses or theliquids into which they are dissolved may, however, easily exceed thetemperature optimum for the carbonic anhydrase used to capture the CO₂.Thus, one of the drawbacks of using enzyme based solutions is thatextensive cooling may be need prior to contacting the CO₂-containinggas/liquid with the carbonic anhydrase, and cooling is an energyconsuming process.

SUMMARY OF THE INVENTION

One aspect of the present invention, is the use of heat-stable carbonicanhydrase of bacterial or archaeal or fungal origin, but excludinggamma-class carbonic anhydrase from Methanosarcina thermophila strainTM-1 (DSM 1825), for extraction of carbon dioxide from a carbondioxide-containing medium. The heat-stable carbonic anhydrase useful inthe present invention maintain activity at temperatures above 45° C. forat least 15 minutes. The heat-stable carbonic anhydrases are inparticular used in a bioreactor capable of extracting CO₂ emitted fromcombustion, or from raw natural gas or a syngas or a biogas. The heatstability is also useful when exposing carbonic anhydrase toenvironments where the temperature can exceed 45° C. during use, orduring idle periods, for example storage in a hot warehouse.

In another aspect, the present invention provides an isolatedpolypeptide having carbonic anhydrase activity at elevated temperatures,selected from the group consisting of:

a) a polypeptide having an amino acid sequence which has at least 94%identity with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4,or at least 91% identity with the amino acid sequence of SEQ ID NO: 6,or at least 96% identity with the amino acid sequence of SEQ ID NO: 8,or at least 87% identity with the amino acid sequence of SEQ ID NO: 10,or at least 97% identity with the amino acid sequence of SEQ ID NO: 12;

b) a polypeptide encoded by a nucleic acid sequence which hybridizesunder medium stringency conditions with:

-   -   i) a polynucleotide sequence encoding a mature polypeptide        selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:        4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12,    -   ii) a polynucleotide sequence selected from the group consisting        of regions of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5, SEQ ID        NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 encoding a mature enzyme,    -   iii) the cDNA sequence contained in a polynucleotide sequence        selected from the group consisting of regions of SEQ ID NO: 1,        SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ        ID NO: 11 encoding a mature enzyme,    -   iv) a subsequence of (i), (ii) or (iii) of at least 100        contiguous nucleotides, or    -   v) a complementary strand of (i), (ii), (iii) or (iv); and

c) a fragment of (a) or (b) having carbonic anhydrase activity.

In a further aspect, the invention provides a composition comprising apolypeptide of the invention and a method for preparing such acomposition comprising admixing the polypeptide of the invention with anexcipient.

In further aspects, the invention provides an isolated polynucleotidehaving a nucleotide sequence which encodes for a polypeptide of theinvention and a nucleic acid construct comprising such a polynucleotideas well as a recombinant vector or recombinant host cell comprising sucha nucleic acid construct.

In a further aspect, the present invention provides a method forproducing the polypeptide of the present invention by cultivating astrain, which in its wild-type form is capable of producing thepolypeptide, or by cultivation of a recombinant host cell comprising arecombinant expression vector coding for a polypeptide of the presentinvention under conditions conducive for production of the polypeptideand recovering the polypeptide.

In a further aspect, the present invention provides a bioreactorsuitable for extracting carbon dioxide.

DRAWINGS

FIG. 1 is a schematic presentation of a hollow fiber contained liquidmembrane bioreactor. The numbers represent the following features: 1.Carbon Dioxide (CO₂) tank; 2. Nitrogen (N₂) or Methane (CH₄) tank; 3.Mass flow controllers (MFC); 4. Membrane liquid reservoir; 5. Liquidpump; 6. Pressure gauge; 7. Hollow fiber membrane bioreactor (module);8. Waste; 9. Feed gas; 10. Scrubbed gas; 11. Mass flow meter (MFM); 12Gas sampling valve; 13. Gas chromatograph; 14. Feed gas in; 15. Scrubbedgas out; 16. Liquid in; 17. Liquid out.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention concerns the use of heat-stablecarbonic anhydrases for the extraction of CO₂ from CO₂-containing media,such as a gas, a liquid or multiphase mixture. The present invention isin particular useful where the temperature of the CO₂-containing mediumis above the temperature optimum for commercially available carbonicanhydrases, such as CA-I or CA-II isolated from human or bovineerythrocytes.

A further aspect of the invention is to provide heat-stable carbonicanhydrases suitable for extracting CO₂ from gas phases or solutions withtemperatures above the temperature optimum for commercially availablecarbonic anhydrases, such as CA-I or CA-II isolated from human or bovineerythrocytes. Heat-stable carbonic anhydrases of the present inventionare preferably of bacterial or archaeal or fungal origin and may be ofany of the distinct CA classes; alpha, beta, gamma or delta, except forthe gamma-class carbonic anhydrase from Methanosarcina thermophila TM-1(DSM 1825). In a preferred embodiment the carbonic anhydrases belong tothe alpha- or beta-class, and more preferred they belong to thealpha-class.

DEFINITIONS

The term “archaeal origin” includes molecules such as polypeptides,nucleic acids, DNA and RNA derived from archaea. It is also intended toinclude modified or mutated molecules where the parent moleculeoriginally was derived from archaea. The origin of the modified ormutated molecule should still be recognizable, preferably polypeptideand nucleic acid sequences are at least 60% identical to the parentmolecule, more preferably it is at least 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% identical to the parent molecule.

The term “bacterial origin” includes molecules such as polypeptides,nucleic acids, DNA and RNA derived from bacteria. It is also intended toinclude modified or mutated molecules where the parent moleculeoriginally was derived from bacteria. The origin of the modified ormutated molecule should still be recognizable, preferably polypeptideand nucleic acid sequences are at least 60% identical to the parentmolecule, more preferably it is at least 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% identical to the parent molecule.

The term “carbonic anhydrase activity” or “CA activity” is definedherein as an EC 4.2.1.1 activity which catalyzes the inter-conversionbetween carbon dioxide and bicarbonate [CO₂+H₂O⇄HCO₃ ⁻+H⁺]. For purposesof the present invention, CA activity is determined according to theprocedure described in Example 3 or 4. One unit of CA activity isdefined after Wilbur [1 U=(1/t_(c))−(1/t_(u))×1000] where U is units andt_(c) and t_(u) represent the time in seconds for the catalyzed anduncatalyzed reaction, respectively (Wilbur, 1948, J. Biol. Chem. 176:147-154). The polypeptides of the present invention have at least 20%,preferably at least 40%, more preferably at least 50%, more preferablyat least 60%, more preferably at least 70%, more preferably at least80%, even more preferably at least 90%, most preferably at least 95%,and even most preferably at least 100% of the CA activity of thepolypeptide consisting of the amino acid sequence of SEQ ID NO: 14.

The term “CO₂-containing medium” is used to describe any material whichmay contain at least 0.001% CO₂, preferably at least 0.01%, morepreferably at least 0.1%, more preferably at least 1%, more preferablyat least 5%, most preferably 10%, even more preferred at least 20%, andeven most preferably at least 50% CO₂. Preferably the CO₂-containingmedium has a temperature between 45° C. and 100° C., more preferablybetween 45° C. and 80° C., even more preferably between 45° C. and 60°C., and most preferably between 45° C. and 55° C. CO₂-containing mediaare in particular gaseous phases, liquids or multiphase mixtures, butmay also be solid. A CO₂-containing gaseous phase is for example rawnatural gas obtainable from oil wells, gas wells, and condensate wells,syngas generated by the gasification of a carbon containing fuel (e.g.,methane) to a gaseous product comprising CO and H₂, or emission streamsfrom combustion processes, e.g., from carbon based electric generationpower plants, or from flue gas stacks from such plants, industrialfurnaces, stoves, ovens, or fireplaces or from airplane or car exhausts.A CO₂-containing gaseous phase may alternatively be from respiratoryprocesses in mammals, living plants and other CO₂ emitting species, inparticular from green-houses. A CO₂-containing gas phase may also beoff-gas, from aerobic or anaerobic fermentation, such as brewing,fermentation to produce useful products such as ethanol, or theproduction of biogas. Such fermentation processes can occur at elevatedtemperatures if they are facilitated by thermophilic microorganisms,this is for example seen in the production of biogas. A CO₂-containinggaseous phase may alternatively be a gaseous phase enriched in CO₂ forthe purpose of use or storage. The above described gaseous phases, mayalso occur as multiphase mixtures, where the gas co-exist with a certaindegree of fluids (e.g., water or other solvents) and/or solid materials(e.g., ash or other particles). CO₂-containing liquids are any solutionor fluid, in particular aqueous liquids, containing measurable amountsof CO₂, preferably at one of the levels mentioned above. CO₂-containingliquids may be obtained by passing a CO₂-containing gas or solid (e.g.,dry ice or soluble carbonate containing salt) into the liquid.CO₂-containing fluids may also be compressed CO₂ liquid (that containscontaminants, such as dry-cleaning fluid), or supercritical CO₂, or CO₂solvent liquids, like ionic liquids.

The term “CO₂ extraction” is to be understood as a reduction of CO₂ froma CO₂-containing medium. Such an extraction may be performed from onemedium to another, e.g., gas to liquid, liquid to gas, gas to liquid togas, liquid to liquid or liquid to solid, but the extraction may also bethe conversion of CO₂ to bicarbonate or carbonate within the samemedium. The term CO₂ capture is also used to indicate extraction of CO₂from one medium to another or conversion of CO₂ to bicarbonate orcarbonate.

When used herein the term “coding sequence” indicates a nucleotidesequence, which directly specifies the amino acid sequence of itsprotein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG. The codingsequence may be a DNA, cDNA, mRNA, or recombinant nucleotide sequence.

The term “functional fragment of a polypeptide” is used to describe apolypeptide which is derived from a longer polypeptide, e.g., a maturepolypeptide, and which has been truncated either in the N-terminalregion or the C-terminal region or in both regions to generate afragment of the parent polypeptide. To be a functional polypeptide thefragment must maintain at least 20%, preferably at least 40%, morepreferably at least 50%, more preferably at least 60%, more preferablyat least 70%, more preferably at least 80%, even more preferably atleast 90%, most preferably at least 95%, and even most preferably atleast 100% of the CA activity of the parent polypeptide.

The term “fungal origin” includes molecules such as polypeptides,nucleic acids, DNA and RNA derived from fungi. It is also intended toinclude modified or mutated molecules where the parent moleculeoriginally was derived from bacteria. The origin of the modified ormutated molecule should still be recognizable, preferably polypeptideand nucleic acid sequences are at least 60% identical to the parentmolecule, more preferably it is at least 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% identical to the parent molecule.

The term “identity” is used to describe the relatedness between twoamino acid sequences or two nucleic acid sequences. For purposes of thepresent invention, the alignment of two amino acid sequences isdetermined by using the Needle program from the EMBOSS package(http://emboss.org) version 2.8.0. The Needle program implements theglobal alignment algorithm described in Needleman and Wunsch, 1970, J.Mol. Biol. 48: 443-453. The substitution matrix used is BLOSUM62, gapopening penalty is 10, and gap extension penalty is 0.5. The degree ofidentity between two amino acid sequences is calculated as the number ofexact matches in an alignment of the two sequences, divided by thelength of the shortest sequence. The result is expressed in percentidentity. An exact match occurs when the “first sequence” and the“second sequence” have identical amino acid residues in the samepositions of the overlap (in the alignment example below this isrepresented by “|”). In the purely hypothetical alignment example below,the overlap is the amino acid sequence “HTWGERNL” of Sequence 1; or theamino acid sequence “HGWGEDANL” of Sequence 2. In the example a gap isindicated by a “-”

The degree of identity between two nucleotide sequences is determinedusing the same algorithm, software package and settings as describedabove.

The term “expression” includes any step involved in the production ofthe polypeptide including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

The term “expression vector” is defined herein as a linear or circularDNA molecule that comprises a polynucleotide encoding a polypeptide ofthe invention, and which is operably linked to additional nucleotidesthat provide for its expression.

The term “heat-stable” or “thermostable” as used herein in reference toan enzyme, such as a carbonic anhydrase, indicates that the enzyme isfunctional or active (i.e., can perform catalysis) at an elevatedtemperature, i.e., above 45° C., preferably above 50° C., morepreferably above 55° C., more preferably above 60° C., even morepreferably above 65° C., most preferably above 70° C., most preferablyabove 80° C., most preferably above 90° C., and even most preferablyabove 100° C. The temperature stability of the carbonic anhydrase can beincreased to some extent by way of formulation, e.g., by immobilizationof the enzyme. In order for an enzyme to be considered as heat-stable itremains active for at least 15 minutes, preferably for at least 2 hours,more preferably for at least 24 hours, more preferably for at least 7days, even more preferably for at least 14 days, most preferably for atleast 30 days, even most preferably for at least 50 days at the elevatedtemperature. Generally, the level of activity is measured after thegiven time at the elevated temperature. The activity may be comparedwith the enzyme activity prior to the temperature elevation. Preferably,the activity is at least 40% after the given time at the elevatedtemperature, more preferably the activity is at least 50% after thegiven time at the elevated temperature, more preferably the activity isat least 60% after the given time at the elevated temperature, even morepreferably the activity is at least 70% after the given time at theelevated temperature, most preferably the activity is at least 80% afterthe given time at the elevated temperature, even most preferably theactivity is at least 90%, and absolutely most preferred the level ofactivity is at least equal to or unchanged after the given time at theelevated temperature.

The term “host cell”, as used herein, includes any cell type which issusceptible to transformation, transfection, transduction, and the likewith a nucleic acid construct comprising a polynucleotide of the presentinvention.

The term “isolated polypeptide” as used herein refers to a polypeptidewhich is at least 20% pure, preferably at least 40% pure, morepreferably at least 60% pure, even more preferably at least 80% pure,most preferably at least 90% pure, and even most preferably at least 95%pure, as determined by SDS-PAGE.

The term “operably linked” denotes herein a configuration in which acontrol sequence is placed at an appropriate position relative to thecoding sequence of the polynucleotide sequence such that the controlsequence directs the expression of the coding sequence of a polypeptide.

The term “region of nucleotide sequence encoding a mature polypeptide”as used herein means the region of a nucleotide sequence counting fromthe triplet encoding the first amino acid of a mature polypeptide to thelast triplet encoding the last amino acid of a mature polypeptide.

The term “polypeptide fragment” is defined herein as a polypeptidehaving one or more amino acids deleted from the amino and/or carboxylterminus of a sequence of the present invention or a homologous sequencethereof, wherein the fragment has CA activity.

The term “secreted polypeptide” as used herein is to be understood as apolypeptide which after expression in a cell is either transported toand released to the surrounding extracellular medium or isassociated/embedded in the cellular membrane so that at least a part ofthe polypeptide is exposed to the surrounding extracellular medium.

The term “substantially pure polypeptide” denotes herein a polypeptidepreparation which contains at most 10%, preferably at most 8%, morepreferably at most 6%, more preferably at most 5%, more preferably atmost 4%, at most 3%, even more preferably at most 2%, most preferably atmost 1%, and even most preferably at most 0.5% by weight of otherpolypeptide material with which it is natively associated. It is,therefore, preferred that the substantially pure polypeptide is at least92% pure, preferably at least 94% pure, more preferably at least 95%pure, more preferably at least 96% pure, more preferably at least 96%pure, more preferably at least 97% pure, more preferably at least 98%pure, even more preferably at least 99%, most preferably at least 99.5%pure, and even most preferably 100% pure by weight of the totalpolypeptide material present in the preparation. The polypeptides of thepresent invention are preferably in a substantially pure form. Inparticular, it is preferred that the polypeptides are in “essentiallypure form”, i.e., that the polypeptide preparation is essentially freeof other polypeptide material with which it is natively associated. Thiscan be accomplished, for example, by preparing the polypeptide by meansof well-known recombinant methods or by classical purification methods.

Herein, the term “substantially pure polypeptide” is synonymous with theterms “isolated polypeptide” and “polypeptide in isolated form”.

The term “Syngas” or “synthesis gas” is used to describe a gas mixturethat contains varying amounts of carbon monoxide and hydrogen generatedby the gasification of a carbon containing fuel (e.g., methane ornatural gas) to a gaseous product with a heating value. CO₂ is producedin the syngas reaction and must be removed to increase the heatingvalue.

The term “thermophilic” in relation to an organism, describes anorganism which thrives at relatively high temperatures, i.e., above 45°C. Hyperthermophilic organisms thrive in extremely hot environments,that is, hotter than around 60° C. with an optimal temperature above 80°C.

Use of Heat-Stable Carbonic Anhydrases

Currently, two heat-stable carbonic anhydrase are known, namely thebeta-class CA (Cab) from Methanobacterium thermoautotrophicum ΔH, whichhas been reported to be heat stable to up to 75° C. (Smith and Ferry,1999, J. Bacteriol. 181: 6247-6253) and the gamma-class carbonicanhydrase (Cam) from Methanosarcina thermophila TM-1. Cam was isolatedfor the first time in 1994 (Alber and Ferry, 1994, Proc. Natl. Acad.Sci. USA 91: 6909-1913), and in 1996 it was shown to be stable toheating at 55° C. for 15 min (Alber and Ferry, 1996, J. Bacteriol. 178:3270-3274). Cam is the only isolated enzyme of the gamma-class, and hasbeen subject to a lot of characterization studies since its discovery.However, it has never been suggested to exploit the thermostability ofthese enzymes in any technical uses. US 2004/0259231 discloses the useof Cam as well as the non-thermostable human CA isoform IV in a CO₂solubilization and concentration process, there is however no indicationthat Cam is preferable over CA-IV.

To our knowledge, no heat-stable alpha-class carbonic anhydrasesisolated from an organism occurring in nature (naturally occurringheat-stable alpha-carbonic anhydrase) have been described until thisday. US 2006/0257990 describes variants of human carbonic anhydrase IIwith a certain degree of thermostability.

One aspect of the present invention is the technical application ofheat-stable carbonic anhydrases in the extraction of CO₂ from aCO₂-containing medium, such as a gas, a liquid, or multiphase mixture.Preferably, the CO₂ is extracted to another medium such as a gas orliquid separated from the first medium, but the extraction may also bethe conversion of CO₂ to bicarbonate within the same medium. The presentinvention is in particular useful where the temperature of theCO₂-containing medium is above the temperature optimum for commerciallyavailable carbonic anhydrases, such as CA-I or CA-II isolated from humanor bovine erythrocytes, which have temperature optimums at approximately37° C.

In one embodiment of the present invention the heat-stable carbonicanhydrase to be applied in the extraction of CO₂ is of bacterial orarchaeal or fungal origin, except for the gamma-class carbonic anhydrasefrom Methanosarcina thermophila TM-1 (DSM 1825). In another embodimentthe carbonic anhydrases to be applied in the extraction of CO₂ may befrom any of the distinct CA classes; alpha, or beta, or gamma,preferably they belong to the alpha- or beta-class.

In another embodiment the carbonic anhydrases to be applied in theextraction of CO₂ belong to the alpha-class, in particular a naturallyoccurring alpha-class carbonic anhydrase is preferred. Other preferredheat-stable carbonic anhydrases for use in the present invention arethose which are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical with a carbonic anhydrase selected fromthe group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16 orfrom Bacillus clausii KSM-K16 (NCBI acc. No. Q5WD44) or from Bacillushalodurans (NCBI acc. No. Q9KFW1). Alpha-class carbonic anhydrases aregenerally monomers, are inhibited by sulfonamides and posses esteraseactivity (human CA-III is an exception, since this isomer is insensitiveto sulfonamides and does not hydrolyze p-nitrophenylacetate). Further,alpha-carbonic anhydrases are identified by their consensus sequencemotif: S-E-[HN]-x-[LIVM]-x(4)-[FYH]-x(2)-E-[LIVMGA]-H-[LIVMFA](2). Thealpha-carbonic anhydrases are generally secreted which is an advantagewhen expression in industrial scale is needed. Further, alpha-classcarbonic anhydrases is the CA-class with the highest turnover of up to10⁵ molecules of CO₂ per second. An enzyme with a high activity isgenerally an advantage, since the amount of enzyme needed may be reducedor the process is more expedite than with a less active enzyme.

In another embodiment the carbonic anhydrases to be applied in theextraction of CO₂ belong to the beta-class. Preferred heat-stablebeta-carbonic anhydrases for use in the present invention are thosewhich are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%_(,) 97%,98%, 99% or 100% identical with a carbonic anhydrase selected from thegroup consisting of beta-carbonic anhydrase from Methanobacteriumthermoautotrophicum ΔH (NCBI acc. No. Q50565), beta-class carbonicanhydrase from Bacillus clausii KSM-K16 (NCBI acc. No.YP_(—)176370/Q5WE01), beta-carbonic anhydrase from Bacillus halodurans(NCBI acc. No. NP_(—)244152/Q9K7S3), and beta-carbonic anhydrases fromAspergillus fumigatus (NCBI acc. NO Q4WPJ0, A4DA32, Q4WQ18 or A4DA31).Beta-class carbonic anhydrase exist as dimers, tetramers, hexamers andoctamers. Generally, beta-carbonic anhydrases are intracellularproteins, and their turnover approximately 2×10⁴ molecules of CO₂ persecond. Some beta-carbonic anhydrases can also be identified by thefollowing consensus sequence motif: C-[SA]-D-S-R-[LIVM]-x-[AP] asdisclosed on the Expasy homepage under prosite documentation numberPDOC00586 (www.expasy.org/cgi-bin/prosite-search-ac?PDOC00586).

In a further embodiment the carbonic anhydrase to be applied in theextraction of CO₂ belong to the gamma-class carbonic anhydrase, exceptfor the carbonic anhydrase from Methanosarcina thermophila strain TM-1(DSM 1825) (Cam) (Alber and Ferry, 1994, Proc. Natl. Acad. Sci. USA 91:6909-6913). Gamma-class carbonic anhydrases are trimeric, 1000-10000fold less sensitive to sulfonamides and do not possess esteraseactivity. Some gamma-carbonic anhydrases are known to be secreted, andtheir turnover is up to 7×10⁴ molecules of CO₂ per second. Generally,the gamma-class carbonic anhydrase is a very diverse group of proteinsthat share the sequence motif characteristic of the left-handed parallelbeta-helix (LβH) fold (Parisi et al., 2000, Molecular Phylogenetics andEvolution 12: 323-334).

In particular carbonic anhydrase, especially heat-stable carbonicanhydrase, may be used for carbon dioxide extraction from CO₂ emissionstreams, e.g., from carbon-based or hydrocarbon-based combustion inelectric generation power plants, or from flue gas stacks from suchplants, industrial furnaces, stoves, ovens, or fireplaces or fromairplane or car exhausts. Carbonic anhydrases, in particular heat-stablecarbonic anhydrases, may also be used to remove CO₂ in the preparationof industrial gases such as acetylene (C₂H₂), carbon monoxide (CO),chlorine (Cl₂), hydrogen (H₂), methane (CH₄), nitrous oxide (N₂O),propane (C₃H₈), sulfur dioxide (SO₂), argon (Ar), nitrogen (N₂), andoxygen (O₂). Carbonic anhydrase can also be used to remove CO₂ from araw natural gas during the processing to natural gas. Removal of CO₂from the raw natural gas will serve to enrich the methane (CH₄) contentin the natural gas, thereby increasing the thermal units/m³. Raw naturalgas is generally obtained from oil wells, gas wells, and condensatewells. Natural gas contains between 3 to 10% CO₂ when obtained fromgeological natural gas reservoirs by conventional methods. Carbonicanhydrase can also be used to purify the natural gas such that it issubstantially free of CO₂, e.g., such that the CO₂ content is below 1%,preferably below 0.5%, 0.2%, 0.1%, 0.05% and most preferably below0.02%. In resemblance to the methane enrichment of natural gases,carbonic anhydrases can also be used to enrich the methane content inbiogases. Biogases will always contain a considerable degree of CO₂,since the bacteria used in the fermentation process produce methane(60-70%) and CO₂ (30-40%). Biogas production may be performed usingmesophilic or thermophilic microorganisms. The process temperatures formesophilic strains is approximately between 25° C. and 40° C.,preferably between 30° C. and 35° C. In this temperature range acarbonic anhydrase may be of bovine or human origin since there are norequirements to thermostability of the enzyme. However, a carbonicanhydrase that tolerates higher temperatures will offer improvedrobustness in actual use and storage related to biogas processesutilizing mesophilic strains. Thermophilic strains allow thefermentation to occur at elevated temperatures, e.g., from 40° C. to 80°C., and preferably from 50° C. to 70° C. and even more preferably from55° C. to 60° C. In such processes a heat-stable carbonic anhydrase isparticularly useful to remove CO₂ from the methane. The presentinvention provides for the use of a carbonic anhydrase to reduce thecarbon dioxide content in a biogas, preferably the CO₂ content isreduced such that it constitutes less than 25%, more preferably lessthan 20%, 15%, 10%, 5%, 2%, 1%, 0.5% and most preferably less than 0.1%.In a preferred embodiment the carbonic anhydrase is heat-stable.Furthermore, carbonic anhydrase may be applied in the production ofsyngas by removing the CO₂ generated by the gasification of a carboncontaining fuel (e.g., methane or natural gas) thereby enriching the CO,H₂ content of the syngas. Where syngas production occurs at elevatedtemperatures the use of a heat-stable carbonic anhydrase is anadvantage. The present invention provides for the use of a carbonicanhydrase to reduce the carbon dioxide content in a syngas production.Preferably, the CO₂ content is reduced such that it constitutes lessthan 25%, more preferably less than 20%, 15%, 10%, 5%, 2%, 1%, 0.5% andmost preferably less than 0.1%. In a preferred embodiment the carbonicanhydrase is heat-stable. Preferably, the carbonic anhydrases to be usedfor CO₂ extraction as described above maintain activity at temperaturesabove 45° C., preferably above 50° C., more preferably above 55° C.,more preferably above 60° C., even more preferably above 65° C., mostpreferably above 70° C., most preferably above 80° C., most preferablyabove 90° C., and even most preferably above 100° C. for at least 15minutes, preferably for at least 2 hours, more preferably for at least24 hours, more preferably for at least 7 days, even more preferably forat least 14 days, most preferably for at least 30 days, even mostpreferably for at least 50 days at the elevated temperature. Thetemperature stability of the carbonic anhydrase can be increased to someextent by way of formulation, e.g., by immobilization of the enzyme.

In an aspect of the present invention the CO₂ extraction from aCO₂-containing medium is performed in enzyme based bioreactors. Beforethe carbon dioxide-containing medium is processed in a bioreactor, itmay be purified to free it from contaminants which may disturb theenzymatic reaction or interfere with bioreactor functionality in otherways, e.g., by clotting outlets or membranes. Gasses/multiphase mixturesemitted from combustion processes, e.g., flue gases or exhausts, arepreferably cleared of ash, particles, NO_(x) and/or SO₂, before thegas/multiphase mixture is passed into the bioreactor. The raw naturalgas from different regions may have different compositions andseparation requirements. Preferably, oil, condensate, water and naturalgas liquids, if present in the raw natural gas, are removed prior to theextraction of CO₂ in an enzyme based bioreactor. The CO₂ from the rawnatural gas may be extracted in the same process as the sulfur removal,or it may be extracted in a completely separate process. If the gas atthis point exceeds the temperature optimum of the carbonic anhydrase ofthe present invention, some degree of cooling may be needed. Preferably,the reaction temperature is between 45° C. and 100° C., more preferablybetween 45° C. and 80° C., even more preferably between 45° C. and 60°C., and most preferably between 45° C. and 55° C. However, due to thethermostability of the enzymes of the present invention, the need forcooling is at least 5° C. less than if a CA-I or CA-II isolated fromhuman or bovine erythrocytes is applied in the bioreactor.

One type of bioreactor useful with the present invention is based on aprocess in which a mixed gas stream (e.g., containing oxygen, nitrogenand carbon dioxide) contacts the enzyme, carbonic anhydrase, at agas-liquid interface to catalyze the conversion of carbon dioxidecontained in the gas to bicarbonate or carbonate. The gas-liquidinterface in such a bioreactor can for example be provided by an enzymebased hollow fiber membrane bioreactor (HFMB). An example of HFMB is ahollow fiber contained liquid membrane (HFCLM) as described by Majumdaret al., 1988, AlChE 1135-1145. CLMs are made by sandwiching a coreliquid between two polymer membranes. The core liquid is preferablycontinuously re-supplied through a reservoir of liquid membrane solvent.An alternative type of enzyme based CLM permeator useful in a bioreactoris described in Cowan et al., 2003, Ann. NY Acad. Sci. 984: 453-469(hereby incorporated by reference). In summary, the bioreactor of thisreference comprise a liquid membrane constructed by sandwiching acarbonic anhydrase containing phosphate buffered solution between twohydrophobic, microporous, polypropylene membranes (e.g., CelgardPP-2400). The CA concentration is preferably between 100-166 micro-M,and the buffer has a phosphate concentration between 50-75 mM and a pHbetween 6.4 and 8.0. Preferred concentrations of CA and of buffer are afunction of the feed CO₂ concentration. The pH optimum is a function ofthe CO₂ concentration and the buffer strength. The thickness of theaqueous phase is preferably 330 micro-m, but may be varied from 70micro-m to 670 micro-m by the use of annular spacers. Preferred membranethickness is determined principally by the desired selectivity towardsother gases such as NO₂ or O₂ and secondarily by desired permeance. Theliquid membrane fluid volume is maintained by hydrostatic fluid additionfrom a reservoir, ensuring a constant liquid membrane thickness andprevents separation between the polymer membrane and the metal support.One side of the CLM (the feed membrane) is contacted with aCO₂-containing feed gas stream, and the other side of the CLM (the sweepmembrane) is in contact with a CO₂-free sweep gas stream, for exampleargon. In this bioreactor CO₂ from the feed gas stream is converted tobicarbonate in the liquid phase and then returned as CO₂ to the sweepgas stream from where it can be stored in the form of compressed CO₂.The entire process is catalysed by the carbonic anhydrase. The CLMpermator described above is capable of capturing CO₂ from feed gasstreams with down to 0.1% CO₂. Alternative CLM permators are composed ofhollow-fiber membrane mats, e.g., Celgard X40-200 or X30-240 instead ofhydrophobic, microporous, polypropylene membranes. The same CAconcentration, buffer concentration and pH can be used with hollow-fiberCLMs. The hollow-fiber permeator can be arranged into different designs.In one design the permeator is arranged much like a heat exchanger andconsists of multiple sets of hollow fiber feed fibers and hollow fibersweep fibers arranged orthogonally while a carrier fluid fills the spacebetween the feed and sweep fiber bundles (see for example Majumdar etal., 1988, AlChE 1135-1145). Another design is a spiral wound hollowfiber design that can operate in either co-current or counter-currentmode. WO 04/104160 describes these and other hollow-fiber permatordesigns in more detail, see in particular FIGS. 1 to 14 (herebyincorporated by reference). WO 04/104160 describes the use of aphosphate buffer as the membrane liquid. When carbonic anhydrase isadded to the membrane liquid it was either dissolved in phosphate bufferor 1 M NaHCO₃.

The present inventors have realized that when using a bicarbonate bufferas the membrane liquid the pH of the buffer is important for the amountof CO₂ that can be extracted from the flue gas. An increase in the pH ofthe bicarbonate solution increases the rate of the hydration of carbondioxide to bicarbonate. In a preferred embodiment of the presentinvention the membrane liquid is a bicarbonate buffer, such as sodiumbicarbonate, potassium bicarbonate, cesium bicarbonate or anothersuitable salt of the bicarbonate. The pH of the bicarbonate buffer ispreferably above 8.5, more preferably above 9.0 and even more preferablyabove 9.5, even more preferred above 9.95, and most preferably above10.5 or above pH 11. The increase of the buffer pH allows for areduction in the amount of carbonic anhydrases needed to extract CO₂from the feed gas. Preferably the amount of carbonic anhydrase is below2 g enzyme protein/L membrane liquid, more preferably it is below 1.5g/L, even more preferably below 1 g/L, even more preferably below 0.6g/L, even more preferably below 0.3 g/L and even more preferably below0.1 g/L, and most preferably below 0.01 g/L, and even most preferablybelow 0.001 g/L.

Another type of bioreactor which may be useful in the present inventionis based on a process in which a gas phase or multiphase mixture, iscontacted with a liquid phase under conditions where the CO₂ in the gasphase is absorbed by the liquid phase where it is converted intobicarbonate by carbonic anhydrase. Preferably, the reaction temperatureis between 45° C. and 100° C., more preferably between 45° C. and 80°C., even more preferably between 45° C. and 60° C., and most preferablybetween 45° C. and 55° C. The bicarbonate enriched liquid is removedfrom the reactor by a continuous flow, to ensure that the equilibriumbetween CO₂ and bicarbonate is shifted towards continuous conversion ofCO₂. The gas phase dissolution into the liquid phase is dependent on thesurface contact area between the gas and liquid. A large contact areacan either be achieved by passing liquid and CO₂-containing gas througha packed column or by bubbling the CO₂-containing gas through the liquidgenerating an elevated pressure in the reaction chamber. Reactors ofthese types are described in U.S. Pat. No. 6,524,843 and WO 2004/007058,respectively; both references are hereby incorporated in their entirety.In summary, packed columns can be composed of packings such as raschigrings, berl saddles, intalox metal, intalox saddles, pall rings. Thepacking materials may be a polymer such as nylon, polystyrene apolyethylene, a ceramic such as silica, or a metal such as aluminium. Inboth reactor types the liquid is continuously exchanged, hence carbonicanhydrase must be retained in the reactor by various means. In thepacked columns the carbonic anhydrase can be immobilized on the packingmaterial (for methods of immobilizing CA, see for example in WO2005/114417). In the “bubbling” reactors the carbonic anhydrase can beentrapped in a porous substrate, for example, an insoluble gel particlesuch as silica, alginate, alginatelchitosane,algnate/carboxymethylcellulose, or the carbonic anhydrase can beimmobilized on a solid packing (as in the packed columns) in suspensionin the liquid, or the carbonic anhydrase can be chemically linked in analbumin or PEG network. When the reactors are in operation an aqueous ororganic solvent enters the reactor at one end, preferably the top, andflows to the other end, preferably the bottom, and the CO₂-containinggas stream (feed gas) enters the reactor at one end, preferably at theopposite end of the solvent (the bottom) and the gas passes through theliquid and exits through a gas outlet at the opposite end (preferably,the top of the reactor). The solvent/liquid that exits the reactor isenriched in bicarbonate and the exit gas is reduced in the CO₂ contentcompared to the feed gas. The bicarbonate containing solution may beprocessed in subsequent reactions for example to generate pure CO₂ orcarbonate precipitates such as CaCO₃. The exit gas may also be subjectedto further rounds of CO₂ extraction. In a preferred embodiment of thepresent invention the reactor liquid is a bicarbonate buffer, such assodium bicarbonate, potassium bicarbonate, cesium bicarbonate or anothersuitable salt of the bicarbonate. The pH of the bicarbonate buffer ispreferably above 8.5, more preferably above 9.0 and even more preferablyabove 9.5, even more preferred above 9.95, and most preferably above10.5 or above pH 11.

A third type of bioreactor which is useful in the present invention isdescribed in U.S. Pat. No. 7,132,090, hereby incorporated by reference.In summary, gaseous CO₂, or CO₂ from a multiphase mixture is diffusedinto a capturing liquid by allowing the gaseous CO₂ to pass through agas diffusion membrane. The CO₂ may pass into the liquid by diffusion(pressure aided) or the transfer may be aided by a carbonic anhydraseimmobilized on the diffusion membrane, e.g., by cross-linking or byaffixing a gel or polymer matrix containing the carbonic anhydrase ontothe diffusion membrane. Since the carbonic anhydrase reacts specificallywith dissolved CO₂, it favors the movement of gaseous CO₂ into the fluidby accelerating the reaction of the dissolved CO₂ and water to formcarbonic acid, thereby removing CO₂ rapidly and allowing the dissolutionof CO₂ from the gas from the feed stream into the water to a greaterextent than it would otherwise. Preferably, the gas diffusion membranehas a high surface area to facilitate a large flow of the gaseous CO₂through the membrane. Suitable membranes include a polypropylene gasexchange membrane, ePTFE (GORE-TEX), Nafion membranes, zeolites,chytosan, polyvinylpyrollindine, cellulose acetate, and immobilizedliquid membranes. The CO₂/bicarbonate rich fluid that emerges from thegas diffusion membrane is passed by a matrix that contains carbonicanhydrase. Preferably, the matrix is contained in a chamber which isseparate from the chamber containing the diffusion membrane. Examples ofsuitable matrixes include beads, fabrics, fibers, membranes,particulates, porous surfaces, rods, and tubes. Specific examples ofsuitable matrixes include alumina, bentonite, biopolymers, calciumcarbonate, calcium phosphate gel, carbon, cellulose, ceramic supports,clay, collagen, glass, hydroxyapatite, ion-exchange resins, kaolin,nylon, phenolic polymers, polyaminostyrene, polyacrylamide,polypropylene, polymerhydrogels, sephadex, sepharose, silica gel, andTEFLON-brand PTFE. The carbonic anhydrase may be immobilized to thematrix or entrapped within it. Once the CO₂ is passed into the liquid anequilibrium between carbonic acid, bicarbonate and carbonate ions willbe established, a process which is catalyzed by carbonic anhydrase. Base(e.g., OH⁻ ions) can then be added to shift the equilibrium to favor theformation of carbonate ions. In the final step, a mineral ion is addedto a solution to precipitate carbonate salts. Alternatively, no base isadded, thereby predominantly generating bicarbonate ion which can beconcentrated using an ion-exchange resin or membrane. The bicarbonatecan then be precipitated using sodium, magnesium or calcium ions. In apreferred embodiment of the present invention the capturing liquid is abicarbonate buffer, such as sodium bicarbonate, potassium bicarbonate,cesium bicarbonate or another suitable salt of the bicarbonate. The pHof the bicarbonate buffer is preferably above 8.5, more preferably above9.0 and even more preferably above 9.5, even more preferred above 9.95,and most preferably above 10.5 or above pH 11. In a preferred embodimentof the present invention the bioreactor operates in steady-stateconditions whereby the CO₂ uptake rate improvement provided by carbonicanhydrase results in overall efficiency improvement of the bioreactor.

The enzyme based bioreactors described above, including a heat-stablecarbonic anhydrase of the present invention, also find moreunconventional applications such as in pilot cockpits, submarinevessels, aquatic gear, safety and firefighting gear and astronaut'sspace suits to keep breathing air free of toxic CO₂ levels. Otherapplications are to remove CO₂ from confined spaces, such as to reducehazardous CO₂ levels from inside breweries and enclosed buildingscarrying out fermentation, and from CO₂ sensitive environments likemuseums and libraries, to prevent excessive CO₂ from causing acid damageto books and artwork.

Carbonic anhydrase can be used as an independent CO₂ extraction catalystor it may alternatively be combined with conventional CO₂ extractiontechnologies such as chemical absorption via amine-based solvents oraqueous ammonia or physical solvents such as Selexol™ (Union Carbide) orpolyethylene glycol ethers. The present inventors have shown that byadding carbonic anhydrase to a MEA solution the efficiency of thescrubbing is significantly increased. In a further embodiment of thepresent invention a carbonic anhydrase, preferably a heat-stablecarbonic anhydrase, is combined with a carbon dioxide absorbing compoundsuch as amine-based compounds such as aqueous alkanolamines includingmonoethanolamine (MEA), diethanolamine (DEA), methyldiethanolamine(MDEA), 2-amino-2-methyl-1-propanol (AMP),2-amino-2-hydroxymethyl-1,3-propanediol (AHPD) or other primary,secondary, tertiary or hindered amine-based solvents, or aqueous saltsof glycine and taurine or other liquid absorbers such as aqueous NaOH,KOH, LiOH, carbonate or bicarbonate solutions at different ionicstrengths or aqueous electrolyte solutions and promoters such aspiperazine, or polyethylene glycol ethers, or a blend of them or analogsor blends thereof. The combination may either be applied in thebioreactors described above or it may be applied to already existing CO₂scrubbing facilities based on more conventional techniques. Inconventional bioreactors, the concentration of alkanolamines istypically 15-30 weight percent. In conventional processes, proprietaryinhibitors, such as Fluor Daniel's EconAmine, are added to provide forincreasing the amine concentration while reducing the risk of corrosion.In the bioreactors described above, the concentration of alkanolaminesis preferably below 15% (V/V), more preferably below 12%, 10%, 8%, 6%,5%, 4%, 3%, 2%, 1%, 0.5%, 0.2% and most preferably below 0.1% (V/V).

Another aspect of the present invention relates to biogas productionwhere the CO₂ extraction is performed directly in the biogasfermentation broth, as an alternative to passing the biogas through abioreactor as described above. By adding carbonic anhydrase to theanaerobic broth, more CO₂ from the gas phase can be converted intobicarbonate, which is the substrate for methane production by themethanogenic Archaea. It has been shown for Methanosarcina thermophilaTM-1 that bicarbonate may be a limiting factor for the methaneproduction, for example cultures of M. thermophila TM-1 grown in lowbicarbonate solution (0.6 mM) showed a considerable lag phase (i.e.,methane production began later) when compared with cultures containingten times higher bicarbonate dosages (6 mM). Additionally, the totalyield of methane was 25 times less at the lower bicarbonate dosage(Murray and Zinder, 1985, Appl. Environ. Microbiol. 50: 49-55).

Another aspect of the present invention is addition of carbonicanhydrase to a fermentation broth, in particular in cases where thebicarbonate concentration in the broth is a limiting factor. Addition ofcarbonic anhydrase can increase the methane production. Particularly,the genus Methanosarcina is frequently present in thermophilic biogasdigesters (Mladenovska and Ahring, 2000, FEMS Microbiol. Ecol. 3:225-229). Hence, a heat-stable carbonic anhydrase will be particularlyuseful if the biogas production is performed at elevated temperaturesusing one or more thermophilic microorganisms, for example methanogenslike Methanosarcina sp. that can use CO₂/biocarbonate as carbon sourcefor growth and methanogenesis.

A further embodiment of the present invention is use of a carbonicanhydrase, in particular a heat-stable carbonic anhydrase, as anadditive in a biogas fermentation broth.

Polypeptides

A polypeptide sequence from Bacillus clausii KSM-K16 similar to thesequences of the present invention is disclosed in the NCBI databaseunder acc. No. Q5WD44 (presented as SEQ ID NO: 14). The sequence istranslated from a nucleotide sequence derived from a genomic sequencingproject on Bacillus clausii KSM-K16, performed by Kao. Based onsimilarity to other alpha-class carbonic anhydrases the nucleotidesequence was assigned to this class, but it has to our knowledge neverbeen expressed and characterized. Hence, the nucleotide sequence wascloned and the polypeptide was expressed for the first time in theexamples of the present application, and it was shown that thepolypeptide possess carbonic anhydrase activity after 15 minutes and 2hours of heating to temperatures above 50° C.

An aspect of the present invention relates to novel heat-stable carbonicanhydrases of the alpha-class type. One embodiment relates to isolatedpolypeptides having an amino acid sequence which has a degree ofidentity of at least 97%, preferably at least 98%, more preferably atleast 99%, most preferably at least 100% to the amino acid sequence ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,or SEQ ID NO: 12, which polypeptide have carbonic anhydrase activity(hereinafter “homologous polypeptides”). In a preferred embodiment, thehomologous polypeptides have an amino acid sequence which differs byseven amino acids, preferably by five amino acids, more preferably byfour amino acids, even more preferably by three amino acids, mostpreferably by two amino acids, and even most preferably by one aminoacid from the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12. Polypeptides withamino acids of position 1 to 237 of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12 are maturepolypeptides of the present invention. Polypeptides with amino acids ofposition 10 to 237 of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, SEQ ID NO: 10, or SEQ ID NO: 12 are recombinant polypeptides ofthe present invention. In a further preferred embodiment the homologouspolypeptides of the present invention have carbonic anhydrase activityat an elevated temperature, i.e., above 45° C., preferably above 50° C.,more preferably above 55° C., more preferably above 60° C., even morepreferably above 65° C., most preferably above 70° C., most preferablyabove 80° C., most preferably above 90° C., and even most preferablyabove 100° C.

A polypeptide of the present invention preferably comprises, morepreferably consists of, amino acids of a mature polypeptide or arecombinant polypeptide selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, and SEQ IDNO: 12 or an allelic variant thereof; or a fragment thereof that hascarbonic anhydrase activity, preferably at an elevated temperature. In apreferred embodiment, a polypeptide comprises, preferably consists of,an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ IDNO: 12. In another preferred embodiment, a polypeptide comprises,preferably consists of, amino acids 1 to 237 or 10 to 237 of an aminoacid sequence selected from the group consisting of SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 or anallelic variant thereof; or a fragment thereof that has carbonicanhydrase activity, preferably at an elevated temperature. In an evenmore preferred embodiment, a polypeptide consists of amino acids 10 to237 of an amino acid sequence selected from the group consisting of SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 andSEQ ID NO: 12 and has the N-terminal amino acid sequence LKASW with aleucine as the most N-terminal amino acid, irrespective of the aminoacid indicated in that position of the respective sequence.

In a further embodiment, the present invention relates to isolatedpolypeptides having carbonic anhydrase activity, preferably at anelevated temperature, which are encoded by polynucleotides whichhybridize under very low stringency conditions, preferably lowstringency conditions, more preferably medium stringency conditions,more preferably medium-high stringency conditions, even more preferablyhigh stringency conditions, and most preferably very high stringencyconditions with:

(i) nucleotides encoding a mature enzyme selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10 and SEQ ID NO: 12,

(ii) a polynucleotide sequence selected from the group consisting ofregions of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ IDNO: 9 and SEQ ID NO: 11 encoding a mature enzyme,

(iii) the cDNA sequence contained in a polynucleotide sequence selectedfrom the group consisting of regions of SEQ ID NO: 1, SEQ ID NO: 3, SEQID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 encoding a matureenzyme,

(iv) a subsequence of (i), (ii) or (iii) of at least 100 contiguousnucleotides, or

(v) a complementary strand of (i), (ii), (iii), or (iv) (Sambrook,Fritsch, and Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2dedition, Cold Spring Harbor, N.Y.).

A subsequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 9, or SEQ ID NO: 11 contains at least 100 contiguousnucleotides or preferably at least 200 contiguous nucleotides. Moreover,the subsequence may encode a polypeptide fragment which has carbonicanhydrase activity, preferably at an elevated temperature.

A polynucleotide sequence selected from the group consisting of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ IDNO: 11 or a subsequence thereof, as well as an amino acid sequenceselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 or a fragmentthereof, may be used to design a nucleic acid probe to identify andclone DNA encoding polypeptides having carbonic anhydrase activity,preferably at an elevated temperature, from an organism expected toencode a carbonic anhydrase, according to methods well known in the art.Carbonic anhydrase producing organisms may be eukaryotes, includingmammals, algae, fungi and plants, prokaryotes including bacterialstrains of different genera or species as well as archaeon. Preferably,such an organism is thermophilic or hyperthermopilic. Even morepreferred the polynucleotide is obtained from a thermophilic Bacillusclausii strain which is not Bacillus clausii KSM-K16. In particular,such probes can be used for hybridization with the genomic or cDNA ofthe genus or species of interest, following standard Southern blottingprocedures, in order to identify and isolate the corresponding genetherein. Such probes can be considerably shorter than the entiresequence, but should be at least 14, preferably at least 25, morepreferably at least 35, and most preferably at least 70 nucleotides inlength. It is, however, preferred that the nucleic acid probe is atleast 100 nucleotides in length. For example, the nucleic acid probe maybe at least 200 nucleotides, preferably at least 300 nucleotides, morepreferably at least 400 nucleotides, or most preferably at least 500nucleotides in length. Both DNA and RNA probes can be used. The probesare typically labeled for detecting the corresponding gene (for example,with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed bythe present invention.

A genomic DNA or cDNA library prepared from such organisms may,therefore, be screened for DNA which hybridizes with the probesdescribed above and which encodes a polypeptide having carbonicanhydrase activity, preferably at an elevated temperature. Genomic orother DNA from such organisms may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA which is homologous with a sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 or a subsequencethereof, the carrier material is used in a Southern blot.

For purposes of the present invention, hybridization indicates that anucleotide sequence hybridizes to a labelled nucleic acid probecorresponding to the nucleotide sequence shown in SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11 itscomplementary strand, or a subsequence thereof, under very low to veryhigh stringency conditions. Molecules to which the nucleic acid probehybridizes under these conditions can be detected using X-ray film.

In a preferred embodiment, the nucleic acid probe is nucleotides 1 to237, nucleotides 238 to 474, nucleotides 475 to 711, of SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO:11. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence which encodes the polypeptide of SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO:12 or a subsequence thereof. In another preferred aspect, the nucleicacid probe is the mature polypeptide coding region of SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micro-g/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures for 12 to 24 hours optimally. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at 45° C. (very low stringency), morepreferably at least at 50° C. (low stringency), more preferably at leastat 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency). In a particular embodiment, the wash is conducted using0.2×SSC, 0.2% SDS preferably at least at 45° C. (very low stringency),more preferably at least at 50° C. (low stringency), more preferably atleast at 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency). In another particular embodiment, the wash is conductedusing 0.1×SSC, 0.2% SDS preferably at least at 45° C. (very lowstringency), more preferably at least at 50° C. (low stringency), morepreferably at least at 55° C. (medium stringency), more preferably atleast at 60° C. (medium-high stringency), even more preferably at leastat 65° C. (high stringency), and most preferably at least at 70° C.(very high stringency).

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at about 5° C. to about10° C. below the calculated T_(m) using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures. The carrier material iswashed once in 6×SCC plus 0.1% SDS for 15 minutes and twice each for 15minutes using 6×SSC at 5° C. to 10° C. below the calculated T_(m).

In another aspect, the present invention relates to artificial variantscomprising a conservative substitution, deletion, and/or insertion ofone or more amino acids has been made to an amino acid sequencecomprising or consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12 or the mature orrecombinant polypeptide thereof. Preferably, amino acid changes are of aminor nature, that is conservative amino acid substitutions orinsertions that do not significantly affect the folding and/or activityof the protein; small deletions, typically of one to about 30 aminoacids; small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue; a small linker peptide of up to about20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain. Examples of conservativesubstitutions are within the group of basic amino acids (arginine,lysine and histidine), acidic amino acids (glutamic acid and asparticacid), polar amino acids (glutamine and asparagine), hydrophobic aminoacids (leucine, isoleucine and valine), aromatic amino acids(phenylalanine, tryptophan and tyrosine), and small amino acids(glycine, alanine, serine, threonine and methionine). Amino acidsubstitutions which do not generally alter specific activity are knownin the art and are described, for example, by Neurath and Hill, 1979,In, The Proteins, Academic Press, New York. The most commonly occurringexchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly. In addition to the 20 standard aminoacids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and alpha-methyl serine) maybe substituted for amino acid residues of a wild-type polypeptide. Alimited number of non-conservative amino acids, amino acids that are notencoded by the genetic code, and unnatural amino acids may besubstituted for amino acid residues. “Unnatural amino acids” have beenmodified after protein synthesis, and/or have a chemical structure intheir side chain(s) different from that of the standard amino acids.Unnatural amino acids can be chemically synthesized, and preferably, arecommercially available, and include pipecolic acid, thiazolidinecarboxylic acid, dehydroproline, 3- and 4-methylproline, and3,3-dimethylproline.

Essential amino acids in the parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (i.e.,carbonic anhydrase activity) to identify amino acid residues that arecritical to the activity of the molecule. See also, Hilton et al., 1996,J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or otherbiological interaction can also be determined by physical analysis ofstructure, as determined by such techniques as nuclear magneticresonance, crystallography, electron diffraction, or photoaffinitylabeling, in conjunction with mutation of putative contact site aminoacids. See, for example, de Vos et al., 1992, Science 255: 306-312;Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992,FEBS Lett. 309: 59-64. A large number of these analyses have alreadybeen performed on carbonic anhydrases, the most important are forexample reviewed in Tripp et al., 2001, J. Biol. Chem. 276: 48615-48618and Lindskog, 1997, Pharmacol. Ther. 74: 1-20. The identities ofessential amino acids can also be inferred from analysis of identitieswith polypeptides which are related to a polypeptide according to theinvention.

Single or multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis, recombination, and/or shuffling, followedby a relevant screening procedure, such as those disclosed byReidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer,1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO95/22625. Other methods that can be used include error-prone PCR, phagedisplay (e.g., Lowman et al., 1991, Biochem. 30:10832-10837; U.S. Pat.No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshireet al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells. Mutagenized DNA molecules thatencode active polypeptides can be recovered from the host cells andrapidly sequenced using standard methods in the art. These methods allowthe rapid determination of the importance of individual amino acidresidues in a polypeptide of interest, and can be applied topolypeptides of unknown structure.

One embodiment of the present invention is an isolated polypeptidehaving carbonic anhydrase activity at elevated temperatures selectedfrom the group consisting of: (a) a polypeptide having an amino acidsequence which has at least 94% identity with the amino acid sequence ofSEQ ID NO: 2 or SEQ ID NO: 4, or at least 91% identity with the aminoacid sequence of SEQ ID NO: 6, or at least 96% identity with the aminoacid sequence of SEQ ID NO: 8, or at least 89% identity with the aminoacid sequence of SEQ ID NO: 10, or at least 97% identity with the aminoacid sequence of SEQ ID NO: 12; (b) a polypeptide encoded by a nucleicacid sequence which hybridizes under medium stringency conditions with:(i) nucleotides encoding a mature polypeptide selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10 and SEQ ID NO: 12, (ii) a polynucleotide sequence selectedfrom the group consisting of regions of SEQ ID NO: 1, SEQ ID NO:3, SEQID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 encoding a matureenzyme, (iii) the cDNA sequence contained in a polynucleotide sequenceselected from the group consisting of regions of SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11encoding a mature enzyme, (iv) a subsequence of (i) or (ii) of at least100 contiguous nucleotides, or (v) a complementary strand of (i), (ii),(iii) or (iv); and (c) a fragment of (a) or (b) having carbonicanhydrase activity.

A particular embodiment of the present invention relates to an isolatedpolypeptide having carbonic anhydrase activity at elevated temperaturesselected from the group consisting of: (a) a polypeptide having an aminoacid sequence which has at least 94%, preferably at least 96%, morepreferred at least 98%, even more preferred at least 99% and mostpreferred at least 100% identity with the amino acid sequence of SEQ IDNO: 2 or SEQ ID NO: 4, or a functional fragment thereof; (b) apolypeptide encoded by a nucleic acid sequence which hybridizes undermedium stringency conditions with: (i) nucleotides encoding a maturepolypeptide of regions of SEQ ID NO: 2 or SEQ ID NO: 4, (ii) apolynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:3 encoding a matureenzyme, (iii) the cDNA sequence contained in a polynucleotide sequenceof regions of SEQ ID NO: 1 or SEQ ID NO: 3 encoding a mature enzyme,(iv) a subsequence of (i) or (ii) of at least 100 contiguousnucleotides, or (v) a complementary strand of (i), (ii), (iii) or (iv);and (c) a fragment of (a) or (b) having carbonic anhydrase activity atelevated temperatures. In a preferred embodiment, the polypeptide has anamino acid sequence which differs by eleven amino acids, preferably bynine amino acids, more preferred by seven amino acids, more preferablyby five amino acids, even more preferably by three amino acids, and mostpreferably by one amino acid from the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 4

Another particular embodiment of the present invention relates to anisolated polypeptide having carbonic anhydrase activity at elevatedtemperatures selected from the group consisting of: (a) a polypeptidehaving an amino acid sequence which has at least 91%, preferably atleast 94%, more preferred at least 96%, even more preferred at least98%, even more preferred at least 99% and most preferred at least 100%identity with the amino acid sequence of SEQ ID NO: 6, or a functionalfragment thereof; (b) a polypeptide encoded by a nucleic acid sequencewhich hybridizes under medium stringency conditions with: (i)nucleotides encoding a mature polypeptide of SEQ ID NO: 6, (ii) apolynucleotide sequence of regions of SEQ ID NO: 5 encoding a matureenzyme, (iii) the cDNA sequence contained in a polynucleotide sequenceof regions of SEQ ID NO: 5 encoding a mature enzyme, (iv) a subsequenceof (i) or (ii) of at least 100 contiguous nucleotides, or (v) acomplementary strand of (i), (ii), (iii) or (iv); and (c) a fragment of(a) or (b) having carbonic anhydrase activity at elevated temperatures.In a preferred embodiment, the polypeptide has an amino acid sequencewhich differs by eleven amino acids, preferably by nine amino acids,more preferred by seven amino acids, more preferably by five aminoacids, even more preferably by three amino acids, and most preferably byone amino acid from the amino acid sequence of SEQ ID NO: 6.

Another particular embodiment of the present invention relates to anisolated polypeptide having carbonic anhydrase activity at elevatedtemperatures selected from the group consisting of: (a) a polypeptidehaving an amino acid sequence which has at least 96%, preferably atleast 97%, more preferred at least 98%, even more preferred at least 99%and most preferred at least 100% identity with the amino acid sequenceof SEQ ID NO: 8, or a functional fragment thereof; (b) a polypeptideencoded by a nucleic acid sequence which hybridizes under mediumstringency conditions with: (i) nucleotides encoding a maturepolypeptide of SEQ ID NO: 8, (ii) a polynucleotide sequence of regionsof SEQ ID NO: 7 encoding a mature enzyme, (iii) the cDNA sequencecontained in a polynucleotide sequence of regions of SEQ ID NO: 7encoding a mature enzyme, (iv) a subsequence of (i) or (ii) of at least100 contiguous nucleotides, or (v) a complementary strand of (i), (ii),(iii) or (iv); and (c) a fragment of (a) or (b) having carbonicanhydrase activity at elevated temperatures. In a preferred embodiment,the polypeptide has an amino acid sequence which differs by eleven aminoacids, preferably by nine amino acids, more preferred by seven aminoacids, more preferably by five amino acids, even more preferably bythree amino acids, and most preferably by one amino acid from the aminoacid sequence of SEQ ID NO: 8.

Another particular embodiment of the present invention relates to anisolated polypeptide having carbonic anhydrase activity at elevatedtemperatures selected from the group consisting of: (a) a polypeptidehaving an amino acid sequence which has at least 89%, preferably atleast 91%, more preferably at least 94%, more preferred at least 96%,even more preferred at least 98%, even more preferred at least 99% andmost preferred at least 100% identity with the amino acid sequence ofSEQ ID NO: 10, or a functional fragment thereof; (b) a polypeptideencoded by a nucleic acid sequence which hybridizes under mediumstringency conditions with: (i) nucleotides encoding a maturepolypeptide of SEQ ID NO: 10, (ii) a polynucleotide sequence of regionsof SEQ ID NO: 9 encoding a mature enzyme, (iii) the cDNA sequencecontained in a polynucleotide sequence of regions of SEQ ID NO: 9encoding a mature enzyme, (iv) a subsequence of (i) or (ii) of at least100 contiguous nucleotides, or (v) a complementary strand of (i), (ii),(iii) or (iv); and (c) a fragment of (a) or (b) having carbonicanhydrase activity at elevated temperatures. In a preferred embodiment,the polypeptide has an amino acid sequence which differs by eleven aminoacids, preferably by nine amino acids, more preferred by seven aminoacids, more preferably by five amino acids, even more preferably bythree amino acids, and most preferably by one amino acid from the aminoacid sequence of SEQ ID NO: 10.

Another particular embodiment of the present invention relates to anisolated polypeptide having carbonic anhydrase activity at elevatedtemperatures selected from the group consisting of: (a) a polypeptidehaving an amino acid sequence which has at least 97%, preferably atleast 97.5%, more preferred at least 98% even more preferred at least99% and most preferred at least 100% identity with the amino acidsequence of SEQ ID NO: 12, or a functional fragment thereof; (b) apolypeptide encoded by a nucleic acid sequence which hybridizes undermedium stringency conditions with: (i) nucleotides encoding a maturepolypeptide of SEQ ID NO: 12, (ii) a polynucleotide sequence of regionsof SEQ ID NO: 11 encoding a mature enzyme, (iii) the cDNA sequencecontained in a polynucleotide sequence of regions of SEQ ID NO: 11encoding a mature enzyme, (iv) a subsequence of (i) or (ii) of at least100 contiguous nucleotides, or (v) a complementary strand of (i), (ii),(iii) or (iv); and (c) a fragment of (a) or (b) having carbonicanhydrase activity at elevated temperatures. In a preferred embodiment,the polypeptide has an amino acid sequence which differs by eleven aminoacids, preferably by nine amino acids, more preferred by seven aminoacids, more preferably by five amino acids, even more preferably bythree amino acids, and most preferably by one amino acid from the aminoacid sequence of SEQ ID NO: 12.

A polypeptide of the invention is an isolated polypeptide, preferablythe preparation of the polypeptide of the invention contains at the most90% by weight of other polypeptide material with which it may benatively associated (lower percentages of other polypeptide material arepreferred, e.g., at the most 80% by weight, at the most 60% by weight,at the most 50% by weight, at the most 40% by weight at the most 30% byweight, at the most 20% by weight, at the most 10% by weight, at themost 9% by weight, at the most 8% by weight, at the most 6% by weight,at the most 5% by weight, at the most 4% by weight at the most 3% byweight, at the most 2% by weight, at the most 1% by weight and at themost 0.5% by weight). Thus, it is preferred that the isolatedpolypeptide of the invention is substantially pure, preferably thepolypeptide is at least 92% pure, i.e., that the polypeptide of theinvention constitutes at least 92% by weight of the total polypeptidematerial present in the preparation, and higher percentages arepreferred such as at least 94% pure, at least 95% pure, at least 96%pure, at least 96% pure, at least 97% pure, at least 98% pure, at least99%, and at the most 99.5% pure. In particular, it is preferred that thepolypeptide of the invention is in “essentially pure form, i.e., thatthe polypeptide preparation is essentially free of other polypeptidematerial with which it is natively associated. This can be accomplished,for example, by preparing the polypeptide of the invention by means ofwell-known recombinant methods.

The polypeptide of the invention may be synthetically made, naturallyoccurring or a combination thereof. In a particular embodiment thepolypeptide of the invention may be obtained from a microorganism suchas a prokaryotic cell, an archaea cell or a eukaryotic cell, inparticular a fungal cell. The cell may further have been modified bygenetic engineering.

Polynucleotides

The present invention also relates to polynucleotides, particularlyisolated polynucleotides, comprising or consisting of a nucleotidesequence encoding a polypeptide of the invention. In a preferred aspecta nucleotide sequence of the present invention is selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 9 and SEQ ID NO: 11. In another preferred aspect, thenucleotide sequence is the mature polypeptide coding region of apolynucleotide selected from the group of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11. The presentinvention also encompasses nucleotide sequences which encode apolypeptide having an amino acid sequence selected from the group of SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 andSEQ ID NO: 12 or a mature polypeptide thereof, which differ from SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ IDNO: 11, respectively, by virtue of the degeneracy of the genetic code.The present invention also relates to subsequences selected from thegroup of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ IDNO: 9 and SEQ ID NO: 11 which encode fragments of SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12,respectively that have carbonic anhydrase activity, preferably at anelevated temperature.

The present invention also relates to mutant polynucleotides comprisingat least one mutation in the mature polypeptide coding sequence selectedfrom the group of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 9 and SEQ ID NO: 11 in which the mutant nucleotidesequence encodes a polypeptide which consists of amino acids 10 to 237of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO:10, or SEQ ID NO: 12, respectively.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides of the present invention from such genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleotidesequence-based amplification (NASBA) may be used. The polynucleotidesmay be cloned from any organism which can be expected to encode acarbonic anhydrase, such organisms may be eukaryotes, including mammals,algae and plants, prokaryotes including bacterial strains of differentgenera or species as well as archaeon. Preferably, the organisms arethermophilic or hyperthermophilic. Even more preferred thepolynucleotide is obtained from a Bacillus clausii strain which is notBacillus clausii KSM-K16.

Modification of a nucleotide sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of a polypeptidewhich comprises an amino acid sequence that has at least onesubstitution, deletion and/or insertion as compared to an amino acidsequence selected from mature polypeptide comprised in SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12.

It will be apparent to those skilled in the art that such modificationscan be made to preserve the function of the enzyme i.e., made outsideregions critical to the function of the enzyme. Amino acid residueswhich are essential to the function are therefore preferably not subjectto modification, such as substitution. Amino acid residues essential tothe function may be identified according to procedures known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis (see,e.g., Cunningham and Wells, 1989, Science 244: 1081-1085). In the lattertechnique, mutations are introduced at every positively charged residuein the molecule, and the resultant mutant molecules are tested forcarbonic anhydrase activity to identify amino acid residues that arecritical to the activity of the molecule. Sites of substrate-enzymeinteraction can be determined by analysis of the three-dimensionalstructure as determined by such techniques as nuclear magnetic resonanceanalysis, crystallography or photoaffinity labeling (see, e.g., de Voset al., 1992, Science 255: 306-312; Smith et al., 1992, Journal ofMolecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309:59-64). Moreover, a nucleotide sequence encoding an enzyme of theinvention may be modified by introduction of nucleotide substitutionswhich do not give rise to another amino acid sequence of the enzymeencoded by the nucleotide sequence, but which correspond to the codonusage of the host organism intended for production of the enzyme. Theintroduction of a mutation into the nucleotide sequence to exchange onenucleotide for another nucleotide may be accomplished by site-directedmutagenesis using any of the methods known in the art. Particularlyuseful is the procedure, which utilizes a super coiled, double strandedDNA vector with an insert of interest and two synthetic primerscontaining the desired mutation. The oligonucleotide primers, eachcomplementary to opposite strands of the vector, extend duringtemperature cycling by means of Pfu DNA polymerase. On incorporation ofthe primers, a mutated plasmid containing staggered nicks is generated.Following temperature cycling, the product is treated with DpnI, whichis specific for methylated and hemimethylated DNA to digest the parentalDNA template and to select for mutation-containing synthesized DNA.Other procedures known in the art may also be used. For a generaldescription of nucleotide substitution, one may consult with, e.g., Fordet al., 1991, Protein Expression and Purification 2: 95-107.

The present invention also relates to isolated polynucleotidescomprising, preferably consisting of, a nucleotide sequence whichencoding a polypeptide of the present invention, which hybridize undervery low stringency conditions, preferably low stringency conditions,more preferably medium stringency conditions, more preferablymedium-high stringency conditions, even more preferably high stringencyconditions, and most preferably very high stringency conditions with apolynucleotide probe selected from the group consisting of:

i) a polynucleotide sequence selected from the group consisting of SEQID NO: 1, SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQID NO: 11,

ii) a cDNA sequence contained in a polynucleotide sequence selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9 and SEQ ID NO: 11, or

iii) a subsequence of (i) or (ii) encoding a secreted mature polypeptidehaving the function of the corresponding mature polypeptides comprisedin SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10and SEQ ID NO: 12; or

iv) a complementary strand of (i), (ii), or (iii) (Sambrook, Fritsch,and Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d edition,Cold Spring Harbor, N.Y.).

As will be understood, details and particulars concerning hybridizationof the nucleotide sequences will be the same or analogous to thehybridization aspects discussed in the section titled “polypeptides ofthe invention herein.

The present invention also encompasses a storage medium suitable for usein an electronic, preferably digital, device comprising information ofthe amino acid sequence of polypeptides of the invention or thenucleotide sequences of the polynucleotide of the invention, inparticular any of the polypeptide or polynucleotide sequences of theinvention in an electronic or digital form, such as binary code or otherdigital code. The storage medium may suitably be a magnetic or opticaldisk and the electronic device a computing device and the informationmay in particular be stored on the storage medium in a digital form.

Recombinant Expression Vectors.

The present invention also relates to recombinant expression vectorscomprising a nucleic acid construct of the invention. Nucleic acidconstructs of the invention comprise an isolated polynucleotide of thepresent invention, preferably operably linked to one or more controlsequences which direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences. Alternatively, a polynucleotide sequence of the presentinvention or a nucleic acid construct comprising the polynucleotidesequence may be inserted into an appropriate vector for expression. Increating the expression vector, the coding sequence is located in thevector so that the coding sequence is operably linked with theappropriate control sequences for expression. The control sequences mayeither be provided by the vector or by the nucleic acid constructinserted into the vector.

The control sequence may be an appropriate promoter sequence, anucleotide sequence which is recognized by a host cell for expression ofa polynucleotide encoding a polypeptide of the present invention. Thepromoter may be any nucleotide sequence which shows transcriptionalactivity in the host cell of choice including mutant, truncated, andhybrid promoters, and may be obtained from genes encoding extracellularor intracellular polypeptides either homologous or heterologous to thehost cell. Such promoters are well known in the art. The controlsequence may also be a suitable transcription terminator sequence, asequence recognized by a host cell to terminate transcription. Theterminator sequence is operably linked to the 3′ terminus of thenucleotide sequence encoding the polypeptide. Any terminator which isfunctional in the host cell of choice may be used in the presentinvention, such terminators are well known in the art. The controlsequence may also be a suitable leader sequence, a nontranslated regionof an mRNA which is important for translation by the host cell. Theleader sequence is operably linked to the 5′ terminus of the nucleotidesequence encoding the polypeptide. Any leader sequence that isfunctional in the host cell of choice may be used in the presentinvention, such leader sequences are well known in the art. The controlsequence may also be a signal peptide coding region that codes for anamino acid sequence linked to the amino terminus of a polypeptide anddirects the encoded polypeptide into the cell's secretory pathway. The5′ end of the coding sequence of the nucleotide sequence may inherentlycontain a signal peptide coding region naturally linked in translationreading frame with the segment of the coding region which encodes thesecreted polypeptide. Alternatively, the 5′ end of the coding sequencemay contain a signal peptide coding region which is foreign to thecoding sequence. The foreign signal peptide coding region may berequired where the coding sequence does not naturally contain a signalpeptide coding region. Alternatively, the foreign signal peptide codingregion may simply replace the natural signal peptide coding region inorder to enhance secretion of the polypeptide. However, any signalpeptide coding region which directs the expressed polypeptide into thesecretory pathway of a host cell of choice may be used in the presentinvention. The control sequence may also be a polyadenylation sequence,a sequence operably linked to the 3′ terminus of the nucleotide sequenceand which, when transcribed, is recognized by the host cell as a signalto add polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention. It may also be desirable to add regulatorysequences which allow the regulation of the expression of thepolypeptide relative to the growth of the host cell. Examples ofregulatory systems are those which cause the expression of the gene tobe turned on or off in response to a chemical or physical stimulus,including the presence of a regulatory compound.

An isolated polynucleotide encoding a polypeptide of the presentinvention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide'ssequence prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotide sequences utilizing recombinant DNA methods arewell known in the art. Further, tags which may aid purification orimmobilization of the polypeptide may be added to the polypeptide. Sucha tag may for example be a polyhistedine tag (His tag). Preferably, thetag located in the N-terminal or C-terminal of the polypeptide, and maybe encoded by the vector. Alternatively, the tag may be locatedinternally in the polypeptide, as long as it does not affect thefunctionality of the polypeptide.

The recombinant expression vector may be any vector (e.g., a plasmid,phagemid, phage or virus) that can be conveniently subjected torecombinant DNA procedures and can bring about the expression of thenucleotide sequence. The choice of the vector will typically depend onthe compatibility of the vector with the host cell into which the vectoris to be introduced.

The vectors may be linear or closed circular plasmids. The vector may bean autonomously replicating vector, i.e., a vector that exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g., a plasmid, an extrachromosomal element, aminichromosome, or an artificial chromosome.

The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers that permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like. Examples of bacterial selectable markers are the dal genesfrom Bacillus subtilis or Bacillus licheniformis, or markers that conferantibiotic resistance such as ampicillin, kanamycin, chloramphenicol ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hygB (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),trpC (anthranilate synthase), as well as equivalents thereof. Preferredfor use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits stable integration of the vector into the host cell'sgenome or autonomous replication of the vector in the cell independentof the genome.

For integration into the host cell genome, the vector may rely on thenucleotide sequence encoding the polypeptide or any other element of thevector for stable integration of the vector into the genome byhomologous or nonhomologous recombination. Alternatively, the vector maycontain additional nucleotide sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleotide sequences enable the vector to be integrated intothe host cell genome at a precise location(s) in the chromosome(s). Toincrease the likelihood of integration at a precise location, theintegrational elements should preferably contain a sufficient number ofnucleotides, such as 100 to 1,500 base pairs, preferably 400 to 1,500base pairs, and most preferably 800 to 1,500 base pairs, which arehighly homologous with the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleotide sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination, or by random integration.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are theorigins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1permitting replication in Bacillus. Examples of origins of replicationfor use in a yeast host cell are the 2 micron origin of replication,ARS1, ARS4, the combination of ARS1 and CEN3, and the combination ofARS4 and CEN6. Examples of origins of replication useful in afilamentous fungal cell are AMA1 and ANSI (Gems et al., 1991, Gene 98:61-67; Cullen et al., 1987, Nucleic Acids Research 15: 9163-9175; WO00/24883). Isolation of the AMA1 gene and construction of plasmids orvectors comprising the gene can be accomplished according to the methodsdisclosed in WO 00/24883. The origin of replication may be one having amutation which makes its functioning temperature-sensitive in the hostcell (see, e.g., Ehrlich, 1978, Proceedings of the National Academy ofSciences USA 75: 1433). The vector may also comprise two or more originsof replication, each origin allowing for replication in a different hostcell, e.g., a bacterial origin and yeast origin.

More than one copy of a nucleotide sequence of the present invention maybe inserted into the host cell to increase production of the geneproduct. An increase in the copy number of the nucleotide sequence canbe obtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the nucleotide sequence where cells containingamplified copies of the selectable marker gene, and thereby additionalcopies of the nucleotide sequence, can be selected for by cultivatingthe cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Recombinant Host Cells.

The present invention also relates to recombinant a host cell comprisingthe nucleic acid construct of the invention, which are advantageouslyused in the recombinant production of the polypeptides. A vectorcomprising a nucleotide sequence of the present invention is introducedinto a host cell so that the vector is maintained as a chromosomalintegrant or as a self-replicating extra-chromosomal vector as describedearlier.

The host cell may be a prokaryote such as bacterial cells, an archaea oran eukaryote such as fungal cells, plant cells, insect cells, ormammalian cells.

Useful prokaryotes are bacterial cells such as gram positive bacteriaincluding, but not limited to, a Bacillus cell, e.g., Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus clausii, Bacillus coagulans, Bacillus halodurans,Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillusmegaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillusthuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans orStreptomyces murinus, or gram negative bacteria such as E. coli andPseudomonas sp. In a preferred embodiment, the bacterial host cell is aBacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, orBacillus subtilis cell. In another preferred embodiment, the Bacilluscell is an alkalophilic Bacillus.

The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278).

In a preferred embodiment, the host cell is a fungal cell. “Fungi asused herein includes the phyla Ascomycota, Basidiomycota,Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In,Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK) as well as the Oomycota(as cited in Hawksworth et al., In, Ainsworth and Bisby's Dictionary ofThe Fungi, 8th edition, 1995, CAB International, University Press,Cambridge, UK, page 171) and all mitosporic fungi (Hawksworth et al.,In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995,CAB International, University Press, Cambridge, UK). In a more preferredembodiment, the fungal host cell is a yeast cell. “Yeast as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport, eds,Soc. App. Bacteriol. Symposium Series No. 9, 1980).

In an even more preferred embodiment, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell. In a most preferred embodiment, the yeast host cell is aSaccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomycesdiastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,Saccharomyces norbensis or Saccharomyces oviformis cell. In another mostpreferred embodiment, the yeast host cell is a Kluyveromyces lactiscell. In another most preferred embodiment, the yeast host cell is aYarrowia lipolytica cell.

In another more preferred embodiment, the fungal host cell is afilamentous fungal cell. “Filamentous fungi include all filamentousforms of the subdivision Eumycota and Oomycota (as defined by Hawksworthet al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK). Thefilamentous fungi are characterized by a mycelial wall composed ofchitin, cellulose, glucan, chitosan, mannan, and other complexpolysaccharides. Vegetative growth is by hyphal elongation and carboncatabolism is obligately aerobic. In contrast, vegetative growth byyeasts such as Saccharomyces cerevisiae is by budding of a unicellularthallus and carbon catabolism may be fermentative. In an even morepreferred embodiment, the filamentous fungal host cell is a cell of aspecies of, but not limited to, Acremonium, Aspergillus, Fusarium,Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia,Tolypocladium, or Trichoderma. In a most preferred embodiment, thefilamentous fungal host cell is an Aspergillus awamori, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus nigeror Aspergillus oryzae cell. In another most preferred embodiment, thefilamentous fungal host cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In an even most preferred embodiment, the filamentousfungal parent cell is a Fusarium venenatum (Nirenberg sp. nov.) cell. Inanother most preferred embodiment, the filamentous fungal host cell is aHumicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthorathermophila, Neurospora crassa, Penicillium purpurogenum, Thielaviaterrestris, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023 andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81: 1470-1474. Suitable methods for transforming Fusarium species aredescribed by Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787.Yeast may be transformed using the procedures described by Becker andGuarente, In Abelson and Simon, editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology 194: 182-187, Academic Press,Inc., New York; Ito et al., 1983, Journal of Bacteriology 153: 163; andHinnen et al., 1978, Proceedings of the National Academy of Sciences USA75: 1920.

A particular embodiment of the present invention is a recombinant hostcell transformed with a polynucleotide selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 11 SEQ ID NO: 13 and SEQ ID NO: 15. Preferably,such a host cell does not contain an inherent carbonic anhydraseencoding gene, or such a gene has been disrupted. Thereby therecombinant carbonic anhydrase is the only carbonic anhydrase producedby a recombinant host cell of the present invention.

Methods for Preparing Carbonic Anhydrase

The present invention also relates to methods for producing a carbonicanhydrase enzyme of the invention comprising (a) cultivating a host cellcomprising a nucleotide sequence encoding a carbonic anhydrase whichstrain is capable of expressing and secreting the carbonic anhydrase and(b) recovering the carbonic anhydrase. In a particular embodiment thehost cell is a wild type Bacillus clausii strain, which inherentlycontain a carbonic anhydrase encoding gene. More preferred the wild typestrain is the Bacillus clausii strain deposited as NCIB 10309. Inanother embodiment the host cell is a recombinant host cell as describedabove.

In these methods of the invention, the cells are cultivated in anutrient medium suitable for production of the enzyme using methodsknown in the art. For example, the cell may be cultivated by shake flaskcultivation, small-scale or large-scale fermentation (includingcontinuous, batch, fed-batch, or solid state fermentations) inlaboratory or industrial fermentors performed in a suitable medium andunder conditions allowing the polypeptide to be expressed and/orisolated. The cultivation takes place in a suitable nutrient mediumcomprising carbon and nitrogen sources and inorganic salts, usingprocedures known in the art. Suitable media are available fromcommercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). As the enzyme is secreted into the nutrient medium, theenzyme can be recovered directly from the medium. If the enzyme is notsecreted, it can be recovered from cell lysates.

The enzyme may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the carbonic anhydrase activity, e.g., the method described by(Wilbur, 1948, J. Biol. Chem. 176: 147-154). The set up is based on thepH change of the assay mixture due to the formation of bicarbonate fromcarbon dioxide as given in equation 1: [CO₂+H₂O⇄HCO₃ ⁻+H⁺]. A particularway of performing this activity assay is described in (Chirica et al.,2001, Biochim. Biophys. Acta 1544: 55-63). Further, the kinetics of thecarbonic anhydrase may be assessed by its capability of cleavingpara-nitrophenol-acetate to nitrophenol and acetate.

The enzyme of the present invention may be purified by a variety ofprocedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, Janson and Ryden, editors, VCH Publishers, NewYork, 1989).

Compositions Comprising Polypeptides and Methods for their Preparation

The invention provides a composition comprising a carbonic anhydrase ofthe present invention and preferably an excipient and a method forpreparing such a composition comprising admixing the polypeptide of theinvention with an excipient.

In a particular embodiment the carbonic anhydrase of the invention isthe major (polypeptide) component of the composition, e.g., amono-component composition. The excipient in this context is to beunderstood as any auxilliary agent or compound used to formulate thecomposition and includes solvent (e.g., water, inorganic salts, fillers,pigments, waxes), carriers, stabilizers, cross-linking agents,adhesives, preservatives, buffers and the like.

The composition may further comprise one or more additional enzymes,such as an aminopeptidase, amylase, carbohydrase, carboxypeptidase,catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, decarboxylase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase, laccase,lipase, mannosidase, monooxygenase, nitrilase, oxidase, pectinolyticenzyme, peptidoglutaminase, peroxidase, phytase, polyphenoloxidase,proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

The compositions may be prepared in accordance with methods known in theart and may be in the form of a liquid or a solid composition. Forinstance, the enzyme composition may be formulated using methods knownto the art of formulating technical enzymes and/or pharmaceuticalproducts, e.g., into coated or uncoated granules or micro-granules. Thepolypeptide of the invention may thus be provided in the form of agranule, preferably a non-dusting granule, a liquid, in particular astabilized liquid, a slurry or a protected polypeptide.

For certain applications, immobilization of the polypeptide may bepreferred. An immobilized enzyme comprises two essential functions,namely the non-catalytic functions that are designed to aid separation(e.g., isolation of catalysts from the application environment, reuse ofthe catalysts and control of the process) and the catalytic functionsthat are designed to convert the target compounds (or substrates) withinthe time and space desired (Cao, Carrier-bound Immobilized Enzymes:Principles, Applications and Design, Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim, Germany, 2005). When an enzyme is immobilized it is madeinsoluble to the target compounds (e.g., substrates) it aids convertingand to the solvents used. An immobilized enzyme product can be separatedfrom the application environment in order to facilitate its reuse, or toreduce the amount of enzyme needed, or to use the enzyme in a processwhere substrate is continuously delivered and product is continuouslyremoved from proximity to the enzyme, which, e.g., reduces enzyme cost.Furthermore, enzymes are often stabilized by immobilization. A processinvolving immobilized enzymes is often continuous, which facilitateseasy process control. The immobilized enzyme can be retained as aheterogeneous catalyst by mechanical means, or by inclusion in adefinite space. The latter can be done by microencapsulation, e.g., insemi permeable membranes or by inclusion in UF systems using, e.g.,hollow fiber modules, etc. Immobilization on porous carriers is alsocommonly used. This includes binding of the enzyme to the carrier, e.g.,by adsorption, complex/ionic/covalent binding, or just simple absorptionof soluble enzyme on the carrier and subsequent removal of solvent.Cross-linking of the enzyme can also be used as a means ofimmobilization. Immobilization of enzyme by inclusion into a carrier isalso industrially applied. (Buchholz et al., Biocatalysts and EnzymeTechnology, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, 2005).Specific methods of immobilizing enzymes such as carbonic anhydraseinclude, but are not limited to, spraying of the enzyme together with aliquid medium comprising a polyfunctional amine and a liquid mediumcomprising a cross-linking agent onto a particulate porous carrier asdescribed in WO 2007/036235 (hereby incorporated by reference), linkingof carbonic anhydrase with a cross-linking agent (e.g., glutaraldehyde)to an ovalbumin layer which in turn adhere to an adhesive layer on apolymeric support as described in WO 2005/114417 (hereby incorporated byreference), or coupling of carbonic anhydrase to a silica carrier asdescribed in U.S. Pat. No. 5,776,741 or to a silane, or a CNBr activatedcarrier surface such as glass, or co-polymerization of carbonicanhydrase with methacrylate on polymer beads as described inBhattacharya et al., 2003, Biotechnol. Appl. Biochem. 38: 111-117(hereby incorporated by reference). In an embodiment of the presentinvention carbonic anhydrase is immobilized on a matrix. The matrix mayfor example be selected from the group beads, fabrics, fibers, hollowfibers, membranes, particulates, porous surfaces, rods, structuredpacking, and tubes. Specific examples of suitable matrices includealumina, bentonite, biopolymers, calcium carbonate, calcium phosphategel, carbon, cellulose, ceramic supports, clay, collagen, glass,hydroxyapatite, ion-exchange resins, kaolin, nylon, phenolic polymers,polyaminostyrene, polyacrylamide, polypropylene, polymerhydrogels,sephadex, sepharose, silica gel, precipitated silica, and TEFLON-brandPTFE. In an embodiment of the present invention carbonic anhydrase isimmobilized on a nylon matrix according to the techniques described inMethods in Enzymology volume XLIV (section in the chapter: ImmobilizedEnzymes, pages 118-134, edited by Klaus Mosbach, Academic Press, NewYork, 1976), hereby incorporated by reference.

The polypeptide to be included in the composition may be stabilized inaccordance with methods known in the art e.g., by stabilizing thepolypeptide in the composition by adding and antioxidant or reducingagent to limit oxidation of the polypeptide or it may be stabilized byadding polymers such as PVP, PVA, PEG, sugars, oligomers,polysaccharides or other suitable polymers known to be beneficial to thestability of polypeptides in solid or liquid compositions. Apreservative, such as Proxel, can be added to extend shelf life orperformance in application.

In a further embodiment the composition of the invention is acomposition applicable in the capture of carbon dioxide.

EXAMPLES Example 1

Cloning and Expression of B. clausii Carbonic Anhydrase in B. subtilis

Carbonic anhydrase sequences were identified by PCR screening on genomicDNA from different Bacillus clausii strains. Genomic DNA from the B.clausii strains was prepared by using the Qiagen Blood DNA kit followingthe manufacturer's protocol.

PCR Screening

PCR (1) was performed in a total volume of 50 microliters, the followingreagents were added, 1 microliter of genomic DNA preparation (template),10 pmol of each of the primers (Bcaf1 and Bcar1), dNTPs and Expandpolymerase in buffer #1 (Roche). The PCR conditions were 94° C. for 2min; 9 cycles of 94° C. for 15 sec; 55° C. for 45 sec; 68° C. for 1 min;followed by 68° C. for 10 min; 4° C. for 20 min and 15° C. until the endof the PCR program.

The primers used for the PCR screening were:

Bcaf1 (SEQ ID NO: 19) gcttctgctgctagtttcctgtca Bcar1 (SEQ ID NO: 20)ataatgaaaaccgatttctctgtcgc

Obtained PCR products (SEQ ID NOs: 1, 3, 5, 7, 9, 11 and 13) had thelength of approx. 700 bp, were size-excluded and sequenced with the sameprimers. The amino acid translations of the PCR products from PCR(1)represent SEQ ID NOs: 2, 4, 6, 8, 10, 12 and 14. The mature nativeenzymes start at position 1 of SEQ ID NOs: 2, 4, 6, 8, 10, 12 and 14.Table 1 below indicates the identity at the polypeptide level betweenthe native enzymes translated from the PCR products.

TABLE 1 Identity matrix SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ IDNO: 10 NO: 6 NO: 2 NO: 4 NO: 14 NO: 12 NO: 8 SEQ ID 100 97 91 92 89 9190 NO: 10 SEQ ID 100 92 93 91 93 92 NO: 6 SEQ ID 100 99 94 96 95 NO: 2SEQ ID 100 94 96 96 NO: 4 SEQ ID 100 97 96 NO: 14 SEQ ID 100 100 NO: 12SEQ ID 100 NO: 8

Generation of PCR Fragment for SOE PCR

PCR (2) was performed with the same parameters as in PCR(1), except thatprimers and template were replaced with 10 pmol of each of the primersblcaTSP and bcl1362rev and 1 ul of purified product from PCR(1) (SEQ 1,7, 9, 11 and 13).

bclaTSP: (SEQ ID NO: 21) cttgctgcctcattctgcagccgcgttgaaagcatcatggtcbcl1362rev: (SEQ ID NO: 22) tccgatccccttttccattctactttaatgataatgaaaaccga

The PCR products had an approximate length of 700 bp and the PCRproducts were purified. The PCR products were suitable for a subsequentSOE PCR fusion reaction (see PCR(3)). Due to the nature of primerbclaTSP the translated amino acid sequence of this PCR (2) product waschanged to LLPHSAAALKASW . . . , where LLPHSAAA represents a fragment ofthe amyL gene (see SOE fusion reaction below) and LKASW represents theN-terminal of the truncated mature peptide obtained by recombinantexpression of the CAs. Hence, the mature recombinant peptide of all thecloned CAs start at position 10 in SEQ ID NOs: 2, 8, 10, 12 and 14 andhas the N-terminal amino acid sequence LKASW with a leucine as the mostN-terminal amino acid, irrespective of the amino acid indicated in thatposition of the respective sequence.

SOE Fusion

In PCR(3) the signal peptide from the alpha-amylase from B.licheniformis (AmyL) was fused by SOE fusion as described in WO 99/43835(hereby incorporated by reference) in frame to the DNA encoding thecarbonic anhydrase that was obtained in PCR (2). The nucleotidefragments obtained from PCR(3) containing the carbonic anhydrase codingsequence were integrated by homologous recombination into the Bacillussubtilis host cell genome. The gene construct was expressed under thecontrol of a triple promoter system (as described in WO 99/43835). Thegene coding for chloramphenicol acetyl-transferase was used as maker (asdescribed in Diderichsen et al., 1993, Plasmid 30: 312-315).

Chloramphenicol resistant transformants were analyzed by DNA sequencingto verify the correct DNA sequence of the construct. One expressionclone for each recombinant sequence was selected, (SEQ ID NOs: 2, 8, 10,12 and 14 starting at position 10 with LKASW).

The individual carbonic anhydrase expression clones were fermented on arotary shaking table in 1 L baffled Erlenmeyer flasks each containing400 ml soy based media supplemented with 34 mg/l chloramphenicol. Theclones were fermented for 4 days at 37° C. The carbonic anhydraseactivity in the culture broth was determined according to Wilbur, 1948,J. Biol. Chem. 176: 147-154 (see Example 4). Alternatively, the carbonicanhydrase activity was measured as esterase activity withpara-nitrophenolacetate as substrate according to Chirica et al., 2001,Biochim. Biophys. Acta 1544(1-2): 55-63 (see Example 5).

Example 2

Cloning of CA from B. halodurans

The CA from B. halodurans (SEQ ID NO: 16) was cloned according toExample 1 with the following modifications. No screening was performed.Instead, the genomic DNA of strain B. halodurans C-125 (JCM9153) wasused as template and primers for PCR(2) were

cahTSP (SEQ ID NO: 23) ctgcctcattctgcagccgcgccttccacagaaccagtcgatcahrev: (SEQ ID NO: 24) tccgatccccttttccattctactctattcagtgatcacgtcat.

Due to the nature of primer cahTSP the translated amino acid sequence ofthis PCR (2) product was changed to LPHSAAAPSTEPVD . . . , where LPHSAAArepresents a fragment of the amyL gene (see SOE fusion reaction below)and PSTEPVD represents the N-terminal of the mature peptide obtained byrecombinant expression of the CAs. The final SOE PCR was done accordingto Example 1.

Example 3 Enzyme Purification

Six recombinant carbonic anhydrases (SEQ ID NOs: 2, 8, 10, 12, and 14(cloned as described in Example 1 and SEQ ID NO: 16 cloned as describedin Example 2) were purified by the same identical procedure: The culturebroth was centrifuged (26.000×g, 20 min) and the supernatant wasfiltered through a Whatman 0.45 micro-m filter. The 0.45 micro-mfiltrate was approx. pH 7 and conductivity was approx. 20 mS/cm. The0.45 micro-m filtrate was transferred to 10 mM HEPES/NaOH, pH 7.0 by G25sephadex chromatography and applied to a 100 ml Q-sepharose FF columnequilibrated in 10 mM HEPES/NaOH, pH 7.0. After washing the column withthe equilibration buffer, bound protein was eluted with a linear NaClgradient (0→0.5 M) over 3 column volumes. Fractions were collectedduring elution and these fractions were tested for carbonic anhydraseactivity (see Example 4). Two peaks with CA activity were identified.N-terminal sequencing revealed that the first elution peak contained asuperoxide dismutase and the second elution peak (peak B) containedcarbonic anhydrase. Peak B was diluted 7× with deionized water andapplied to a 40 ml SOURCE 30Q column equilibrated in 10 mM HEPES/NaOH,pH 7.0. The column was washed with equilibration buffer and eluted witha linear NaCl gradient (0→0.5 M). Elution fractions from the column wereanalyzed for CA activity and the positive fractions were analyzed bySDS-PAGE. Fractions which revealed a predominant band on a coomassiestained SDS-PAGE gel were pooled into a carbonic anhydrase batch. Theenzyme purity of CAs corresponding to SEQ ID NOs: 2, 8, 10 and 12 wasestimated to be 80% pure, and the enzyme corresponding to SEQ ID NOs: 14and 16 was above 95% pure.

Example 4 Detection of Carbonic Anhydrase Activity

The test for the detection of carbonic anhydrase was described byWilbur, 1948, J. Biol. Chem. 176: 147-154. The set up is based on the pHchange of the assay mixture due to the formation of bicarbonate fromcarbon dioxide as given in equation 1: [CO₂+H₂O→HCO₃ ⁻+H⁺].

The activity assay used in this study was derived from the procedure ofChirica et al., 2001, Biochim. Biophys. Acta 1544(1-2): 55-63. Asolution containing approximately 60 to 70 mM CO₂ was prepared bybubbling CO₂ into 100 ml distilled water using the tip of a syringeapproximately 45 min to 1 h prior to the assay. The CO₂ solution waschilled in an ice-water bath. To test for the presence of carbonicanhydrase, 2 ml of 25 mM Tris, pH 8.3 (containing sufficient bromothymolblue to give a distinct and visible blue color) were added to two 13×100mm test tubes chilled in an ice bath. To one tube, 10 to 50 microlitersof the enzyme containing solution (e.g., culture broth or purifiedenzyme) was added, and an equivalent amount of buffer was added to thesecond tube to serve as a control. Using a 2 ml syringe and a longcannula, 2 ml of CO₂ solution was added very quickly and smoothly to thebottom of each tube. Simultaneously with the addition of the CO₂solution, a stopwatch was started. The time required for the solution tochange from blue to yellow was recorded (transition point of bromothymolblue is pH 6-7.6). The production of hydrogen ions during the CO₂hydration reaction lowers the pH of the solution until the colortransition point of the bromothymol blue is reached. The time requiredfor the color change is inversely related to the quantity of carbonicanhydrase present in the sample. The tubes must remain immersed in theice bath for the duration of the assay for results to be reproducible.Typically, the uncatalyzed reaction (the control) takes approximately 2min for the color change to occur, whereas the enzyme catalyzed reactionis complete in 5 to 15 s, depending upon the amount of enzyme added.Detecting the color change is somewhat subjective but the error fortriple measurements was in the range of 0 to 1 sec difference for thecatalyzed reaction. One unit is defined after Wilbur [1U=(1/t_(c))-(1/t_(u))×1000] where U is units and t_(c) and t_(u)represent the time in seconds for the catalyzed and uncatalyzedreaction, respectively (Wilbur, 1948, J. Biol. Chem. 176: 147-154).These units are also termed Wilbur-Anderson units (WAU).

Example 5

Kinetic Assay for Carbonic Anhydrase Activity with p-Nitrophenyl Acetate

Twenty microliters purified CA enzyme sample obtained as described inExample 3 (diluted in 0.01% Triton X-100) was placed in the bottom of amicro-titer plate (MTP) well. The assay was started at room temperatureby adding 200 microliters para-nitrophenol-acetate ((pNp-acetate, Sigma,N-8130) substrate solution in the MTP well. The substrate solution wasprepared immediately before the assay by mixing 100 microliterspNP-acetate stock solution (50 mg/ml pNP-acetate in DMSO. Stored frozen)with 4500 microliters assay buffer (0.1 M Tris/HCl, pH 8.0). Theincrease in OD₄₀₅ was monitored. In the assay a buffer blind (20microliters assay buffer instead of CA sample) was included. Thedifference in OD₄₀₅ increase between the sample and the buffer blind wasa measure of the carbonic anhydrase activity (CAactivity=ΔOD₄₀₅(sample)−ΔOD₄₀₅(buffer)).

Example 6 Temperature Stability Assay

The purified CA enzyme (SEQ ID NOs: 2, 8, 10, 12, 14 and 16 obtained asdescribed in Example 3) was diluted 10× in 50 mM HEPES/NaOH, pH 7.5 andaliquots were incubated for 15 minutes at different temperatures (15 to80° C.). CA enzyme of SEQ ID NO: 14 was additionally incubated for 2hours at different temperatures. After incubation, residual activity wasmeasured as described in Example 5. The result of the temperaturestability assay is shown in Table 2. Clearly, CAs from M. thermophila,B. halodurans and B. clausii showed higher thermostability than HumanCAII. Further, B. clausii CA was superior in terms of thermostabilityover the M. thermophila CA. The data for Human CAII and M. thermophilawere taken from Alber and Ferry, 1996, J. Bacteriol. 178: 3270-3274.

TABLE 2 Temperature stability of different carbonic anhydrasesTemperature [° C.] CA 15 25 37 50 55 60 65 70 75 80 Data from Alber &Ferry, 1996, J. Bacteriol. 178: 3270-3274 Residual Human CAII — — 95 7835 5 0 0 0 0 activity [%] M. thermophila — — — — 95 90 80 32 5 0Incubation time 15 min B. halodurans 93 101 105 61 n.d. 15 n.d. 11 n.d.8 (SEQ ID NO 16) B. clausii 92 104 104 107 n.d. 94 n.d. 49 n.d. 43 (SEQID NO 14) B. clausii 99 95 106 104 n.d. 90 n.d. 9 n.d. 14 (SEQ ID NO 2)B. clausii 98 99 102 99 n.d. 93 n.d. 67 n.d. 49 (SEQ ID NO 8) B. clausii101 98 101 89 n.d. 64 n.d. 20 n.d. 28 (SEQ ID NO 10) B. clausii 94 101105 95 n.d. 89 n.d. 63 n.d. 54 (SEQ ID NO 12) Incubation time 2 hours B.clausii 96 100 105 96 n.d. 50 n.d. 37 n.d. 30 (SEQ ID NO 14) n.d. = notdetermined

Differential Scanning Calorimetry (DSC)

The purified CA enzyme (SEQ ID NO: 14 and SEQ ID NO: 16 obtained asdescribed in Example 3) was diluted to approx. 1 mg/ml in 50 mMHEPES/NaOH, pH 7.5. DSC was performed with a 90° C./hour scan rate and20° C. to 90° C. scan range. The melting point of B. clausii (SEQ ID NO:14) and B. halodurans (SEQ ID NO: 16) CA was 67.4° C. and 63.1° C.,respectively.

Example 7 Amino Terminal Protein Sequencing

Purified recombinant CAs (SEQ ID NOs: 2, 8, 10, 12, 14 and 16 obtainedas described in Example 3) were sequenced by Edman sequencing. Thedetermined sequences are shown in Table 3. All sequences match thepredicted mature peptide sequence, except for the CA from B. halodurans(SEQ ID NO: 16) where a truncated enzyme starting at position 18 in SEQID NO: 16 was obtained after purification. The protein molecular weightof the recombinant CAs was determined by Electrospray IonizationTime-Of-Flight Mass Spectrometry (ES-TOF MS).

TABLE 3 N-terminal sequences of recombinant CAs. SEQ ID Molecular weightNO Protein Sequence (Edman) by MS 2 LKASWSYEGE (SEQ ID NO: 25) 25551 Da8 LKASWSYEGD (SEQ ID NO: 26) 25522 Da 10 LKASWSYEGE (SEQ ID NO: 27)25286 Da 12 LKASWSYEGD (SEQ ID NO: 28) 25566 Da 14LKASWSYE (SEQ ID NO: 29) 25628 Da 16 GGAHEVHWSY (SEQ ID NO: 30) 26143 Da

Example 8 Thermal Stability Using Wilbur-Anderson Assay

The thermal stability of purified CA enzymes corresponding to SEQ IDNOs: 2, 8, 10, 12 and 14 and Bovine carbonic anhydrase (Sigma, catalognr. C3934) was measured. The CA's were obtained as described in Example3.

The thermal stability was measured as follows: 10 microliters of eachenzyme was diluted 10 folds in 1 M NaHCO₃ Solution (pH=8.05) and wasincubated for 15 minutes or 2 hours at desired temperature. 1 M NaHCO₃solutions were also heated at the same temperature as control. Solutionswere cooled down to room temperature before conducting the assay. TheWilbur-Anderson activity of the heated enzyme solutions was measuredaccording to the procedure of Example 4 with the following minorchanges. The CO₂ solution was prepared 30 min prior to the assay, theice bath was substituted with a water bath of 4° C., the amount ofenzyme was 10 microliters and the uncatalyzed reaction (the control)takes approximately 40 to 50 seconds. The residual activity afterincubation at elevated temperatures is presented in Table 4.

TABLE 4 Temperature stability of different carbonic anhydrasesTemperature [° C.] CA 25 37 50 60 70 80 Incubation time 15 min ResidualB. clausii 100 125.7 121.5 117.6 35.3 5.4 activity (SEQ ID NO 2) [%] B.clausii 100 106 112.3 109.5 110 16.9 (SEQ ID NO 8) B. clausii 100 89.789.7 86.3 38.0 14.2 (SEQ ID NO 10) B. clausii 100 115.1 125.2 139.6 25.7n.d. (SEQ ID NO 12) B. clausii 100 119.5 99.4 84.9 47.2 6.8 (SEQ ID NO14) Bovine CA 100 100 93.7 4.4 1.1 Incubation time 2 h B. clausii n.d.n.d. n.d. n.d. 43.3 n.d. (SEQ ID NO 8) n.d. = not determined

Example 9

Extraction of CO₂ from a Mixed Gas Stream in a Hollow Fiber Bioreactor

A lab-scale hollow fiber contained liquid membrane bioreactor (HFB) wasset up to selectively capture CO₂ from a gas stream which could resemblea flue gas.

Hollow Fiber Membrane Bioreactor Set-Up

Porous hydrophobic hollow fiber membranes provide a high surface area ofcontact between the gas stream and membrane liquid. As a result theyfacilitate carbonation of a liquid or removal of CO₂ from a liquid. Theselected module consists of 2300 parallel hollow fibers with 0.18 m²active surface area and average pore size of 0.01×0.04 micro-m((Liquicel® MiniModule® 1×5.5 purchased from Membrana, N.C., USA). Thesemembranes are easy to scale-up to industrial scale and have been used inindustry for wastewater treatment and beverage carbonation. A schematicdrawing of the bioreactor set-up is shown in FIG. 1. In the set-upmembrane liquid was passed through the hollow fibers lumen using apositive displacement pump. The liquid flow rate was set to about 2ml/min. The gas stream containing a mixture of 15% CO₂ (9 CubicCentimeters per Minute (CCM)) and 85% N₂ (51 CCM) (feed gas) entered thefeed side of the hollow fibers counter-currently and the treated gasstream (scrubbed gas) exited the module at the sweep side of the hollowfibers. Two mass flow controllers were used to mix nitrogen and carbondioxide with consistent concentration through out the experiments. Amass flow meter was used to monitor the flow of the scrubbed gas and thefeed gas as they exit the reactor. The gas and liquid flows andpressures were adjusted to avoid entering liquid to the gas phase andgas bubbles in the liquid phase of the module.

The purpose of this set-up was the hydration of CO₂ to bicarbonate whichwas measured by analyzing the CO₂ concentration in feed gas and scrubbedgas using a gas chromatograph (GC).

Gas Chromatography Method (GC-TCD)

A Shimadzu 2010 gas chromatograph with a thermal conductivity detectorand a gas sampling valve was used for CO₂ concentration measurement. Acapillary Carboxen Plot 1010 column was used to detect nitrogen andcarbon dioxide. The column was heated isothermally for 7 minutes at 35°C., the temperature was increased with 20° C./min rate to 200° C. and itwas maintained at 200° C. for 2 minutes. Injector and detectortemperatures were maintained at 230° C. Column flow is 1 ml/min, splitratio 10 to 1 and carrier gas was helium. Nitrogen and carbon dioxidepeaks were detected at retention times 6.4 and 15.3 minutes,respectively. The CO₂ peak was calibrated using three carbon dioxidestandards with 1000 ppm, 1% and 10% CO₂ in nitrogen purchased from ScottSpecialty gases (Pennsylvania, USA).

Membrane Liquid

Initially 1 M Sodium bicarbonate pH=8 was selected as membrane liquid.However, it was found that the sodium bicarbonate solution was saturatedwith CO₂ at this pH, as a result it was not a very suitable membraneliquid for the hydration reaction (carbonation). A 1 M sodiumbicarbonate solution with a pH of 9 or above was more suitable for CO₂hydration, since it was not saturated with carbon dioxide/bicarbonate. A1 M sodium bicarbonate solution, pH 9.0 was used as a control solutionwithout enzyme. In another experiment after rinsing the hollow fibermodule with de-ionized (DI) water, a solution of 8 parts 1 M sodiumbicarbonate pH 9.6+2 parts carbonic anhydrase of SEQ ID NO: 14,corresponding to a final CA concentration of 0.6 g pure enzymeprotein/L, was used as membrane liquid. The CO₂ concentration in thefeed gas and scrubbed gas using these membrane liquids was analyzed byGC. Each experiment was at least repeated three times using differentmodules and at least three injections to GC were made.

Results

Table 5 shows the data collected using each membrane liquid. It wasfound that the carbon dioxide concentration in the scrubbed gas exitingthe HFCLMB is highly dependent on the pH of the sodium bicarbonatecontrol solution in the membrane liquid. An increase in the pH of thebicarbonate solution increases the rate of the hydration of carbondioxide to bicarbonate.

Furthermore, it was found that when carbonic anhydrase of SEQ ID NO: 14was added to the sodium bicarbonate solution at room temperature, theamount of CO₂ in the scrubbed gas was significantly reduced and theselectivity of the reactor for CO₂ has been increased substantially. Anenzyme-bicarbonate solution with pH 9.95 was also tested as the membraneliquid and no CO₂ peak was detected in the scrubbed gas. In other words,at pH 9.95 nearly complete removal of CO₂ from feed gas was observed.

TABLE 5 Effect of membrane liquid on the CO₂ concentration of the gasstream exiting the hollow fiber membrane bioreactor % CO2 in % CO2 infeed % CO2 Scrubbed gas gas removed Membrane liquid (avg.) (avg.) (avg.)DI Water 13.6 14.2 4.8 1M NaHCO₃ pH 9.0 11.6 15.0 22.7 1M NaHCO₃ pH 9.510.2 15.3 33.1 0.6 g/L CA in 1M NaHCO₃ 0.85 14.3 94.1 pH 9.5 0.6 g/L CAin 1M NaHCO₃ <0.1 15.3 >99 pH 9.95 These results indicate that carbonicanhydrase of SEQ ID NO: 14 even in low dose (~0.6 g enzyme protein/L)significantly increases the efficiency of the hollow fiber membranereactor when compared to the control.

Example 10

Extraction of CO₂ from a Mixed Gas Stream in a Hollow Fiber CLMBioreactor

A lab-scale hollow fiber contained liquid membrane bioreactor (HFB) wasset up to selectively capture CO₂ from a gas stream which could resemblea biogas composition.

The bioreactor set-up was essentially the same as described in Example9. Except that the gas stream contained a mixture of 40% CO₂ (8 CCM) and60% CH₄ (12 CCM) which entered the feed side of the hollow fibers. Thegas which exits the hollow fiber membrane is termed the enriched gas,since the purpose of this set-up is to show that the percentage ofmethane in a biogas stream can be increased using a carbonic anhydrasecontaining bioreactor which captures CO₂ from the produced gas stream.This is possible by selective hydration of CO₂ component of the gas mixto bicarbonate ions in liquid phase. The efficiency of thisafter-treatment was measured by analyzing the methane concentration infeed gas and enriched gas using a gas chromatograph (GC).

Gas Chromatography Method (GC-FID)

A Shimadzu 2010 gas chromatograph with flame ionization detector and agas sampling valve was used for CH₄ concentration measurement. Acapillary Carboxen Plot 1010 column was used to detect methane. Thecolumn was heated isothermally for 3.5 minutes at 200° C. Injector anddetector temperatures were maintained at 230° C. Column flow was 2.35ml/min, split ratio 20 to 1 and carrier gas was helium. Hydrogen and airflow were 45 and 450 mL/min, respectively. The methane peak was detectedat retention time 1.9 minutes. The CH₄ peak was calibrated using fourmethane standards with 1000 ppm, 1%, 10% and 99% methane in nitrogenpurchased from Scott Specialty gases.

Membrane Liquid

The membrane liquid used was a 1 M Sodium bicarbonate solution at pH 9.3for the control. The carbonic anhydrase enzyme and concentration were asdescribed in Example 9.

Results

Table 6 shows the data collected using CO₂—CH₄ mixtures. From this itcan be seen that at room temperature, the amount of CO₂ removed from thegas stream was increased substantially when a carbonic anhydrase wasadded to the membrane liquid. Therefore, the amount of CO₂ captured inthe bioreactor was significantly increased and as a result, the methanecontent in the exit gas stream was significantly increased.

TABLE 6 Effect of membrane liquid on the methane concentration of thebiogas stream exiting the hollow fiber membrane bioreactor % CH₄ inEnriched % CH₄ in feed % stream stream CO₂ removed Membrane liquid(avg.) (avg.) (avg.) DI Water 62.9 59.4 ~9 1M NaHCO₃ pH 9.3 83.0 59.4~59 0.6 g/L CA in 1M NaHCO₃ 95.7 59.4 ~90 pH 9.3

Example 11

Extraction of CO₂ from a Mixed Gas Stream in a Hollow Fiber MembraneBioreactor Containing MEA in the Membrane Liquid

The present experiment illustrates the effect of adding carbonicanhydrase to a conventional carbon dioxide absorber.

The bioreactor set-up was essentially the same as described in Example9. Except that the gas stream contained a mixture of 28.6% CO₂ (20 CCM)and 71.4% N₂ (50 CCM) which entered the feed side of the hollow fibers.The gas chromatography method was identical to Example 9.

Membrane Liquid

The control membrane liquid used was a monoethanol amine solution (MEA)in water (1% V/V). This was compared with a membrane liquid composed ofa MEA-CA aqueous solution containing 10 parts CA and 1 part MEA and 89parts water, corresponding to a final CA concentration of 0.3 g pureenzyme protein/L of the solution.

Results

The data are presented in Table 7. In summary a HFB with 1% MEA solutioncould remove 48.6% of the total CO₂ in the feed gas. Addition of 0.3 g/Lcarbonic anhydrase significantly increased CO₂ removal in a 1% MEAsolution to 84.3%.

This shows that the carbonic anhydrase of SEQ ID NO: 14 is active inpresence of MEA and can significantly improve the absorption of CO₂ inan MEA-containing liquid. Surprisingly, only a low amount of MEA isneeded in the solution to achieve a high level of CO₂ removal when CA ispresent, compared to what is known in the art. Typical aqueousamine-based CO₂ absorber solutions contain in the range 15-30% amine.

TABLE 7 Effect of membrane liquid on the CO₂ concentration of the gasstream exiting the hollow fiber membrane bioreactor Flue gas % CO₂ inmix Membrane liquid Scrubbed % CO₂ % CO₂ % N₂ Content (V/V) pH gasremoved 28.6 71.4 MEA 1% 11.25 14.7 48.6 28.6 71.4 0.3 g/L CA in 1% MEA10.7 4.5 84.3

1. A method of using a heat-stable carbonic anhydrase for extraction ofcarbon dioxide from a carbon dioxide-containing medium, comprisingcontacting a carbon dioxide-containing medium with a heat stablecarbonic anhydrase that maintains activity at temperatures above 45° C.for at least 15 minutes, wherein the heat stable carbonic anhydrase isan alpha-carbonic anhydrase which is at least 85% identical to thealpha-carbonic anhydrase from Bacillus halodurans (SEQ ID NO: 16),wherein carbon dioxide is extracted from the carbon dioxide-containingmedium.
 2. The method in accordance with claim 1, wherein the carbonicanhydrase is the alpha-carbonic anhydrase from Bacillus halodurans (SEQID NO: 16).
 3. The method of claim 1, where the carbonic anhydrasemaintains activity at temperatures above 45° C. for at least two hours.4. The method of claim 1, where the carbonic anhydrase is used in abioreactor.
 5. The method of claim 4, wherein the bioreactor comprises acontained liquid membrane.
 6. The method of claim 5, wherein themembrane liquid of the contained liquid membrane contains a bicarbonatebuffer with a pH of at least 9.0.
 7. The method of claim 1, wherein thecarbon dioxide-containing medium is a carbonic dioxide-containing gas.8. The method of claim 1, wherein the carbon dioxide-containing mediumis a multiphase mixture.
 9. The method of claim 7, where the carbonicdioxide-containing gas is emitted from combustion or fermentation. 10.The method of claim 7, where the carbonic dioxide-containing gas is aflue gas.
 11. The method of claim 7, where the carbonicdioxide-containing gas is a raw natural gas or a syngas.
 12. The methodof claim 7, where the carbonic dioxide-containing gas is a biogas. 13.The method of claim 1, where the carbon dioxide-containing medium is aliquid.
 14. The method of claim 1, wherein the extraction of carbondioxide is performed at temperatures between 45° C. and 60° C.
 15. Themethod of claim 1, wherein the carbonic anhydrase is an alpha-carbonicanhydrase that is at least 90% identical to the alpha-carbonic anhydrasefrom Bacillus halodurans (SEQ ID NO: 16).
 16. The method of claim 1,wherein the carbonic anhydrase is an alpha-carbonic anhydrase that is atleast 95% identical to the alpha-carbonic anhydrase from Bacillushalodurans (SEQ ID NO: 16).
 17. The method of claim 1, wherein thecarbonic anhydrase is an alpha-carbonic anhydrase that is at least 98%identical to the alpha-carbonic anhydrase from Bacillus halodurans (SEQID NO: 16).